Scaffolds to treat solid tumor cells and escape variants

ABSTRACT

Implantable scaffolds that treat solid tumors and escape variants and that provide effective vaccinations against cancer recurrence are described. The scaffolds include genetically-reprogrammed lymphocytes and a lymphocyte activating moiety.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/437,572 filed on Dec. 21, 2016, the entire contents of which areincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA181413 awardedby the National Institutes of Health. The government has certain rightsin the invention.

REFERENCE TO SEQUENCE LISTING

A computer readable text file, entitled “1QJ8796.txt (SequenceListing.txt)” created on or about Nov. 30, 2017, with a file size of 50KB, contains the sequence listing for this application and is herebyincorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure provides implantable scaffolds that treat solidtumors and escape variants and provide effective vaccinations againstcancer recurrence. The scaffolds include genetically-reprogrammedlymphocytes and a lymphocyte-activating moiety.

BACKGROUND OF THE DISCLOSURE

Cancer immunotherapy describes a field in which a patient's immunesystem is used to treat cancer. For example, cancer immunotherapyincludes the use of vaccines to immunize patients against thedevelopment of cancer. Unfortunately, the responses cancer vaccines canelicit can require months to mature and are usually insufficient tocontrol advanced disease.

Attempts have also been made to more quickly or potently stimulate apatient's own T cells to attack cancer cells. The repertoire ofreceptors normally expressed by T cells generally has a low affinity toself/tumor antigens, however, so this approach has not achievedsufficient success in the fight against cancer.

An emerging immunotherapy approach involves the alteration ofpatient-derived lymphocytes (e.g., T cells) with genes encoding chimericantigen receptors (CARs) that are engineered to have high affinity forselected macromolecular targets in the tumors. The introduced genes canalso produce costimulatory signals to elicit robust T cell expansion.The method involves retrieval of T cells from a patient and redirectingthem ex vivo to express CARs composed of a tumor-specific single-chainantibody (scFv) fused to costimulatory and CD3 signaling domains, whichenable the programmed cells to lyse tumor targets in a human leukocyteantigen (HLA)-independent fashion.

Therapies that employ such CAR-programmed T cells have consistentlyproduced positive results in patients with hematologic malignancies(i.e., liquid blood cancers) in clinical trials. However, when it comesto solid tumors, the effectiveness of these therapies has been limitedby: 1) inefficient homing of the lymphocytes to the tumor site; 2) theimmunosuppressive microenvironment solid tumors create; and 3) thephenotypic diversity of solid tumors which means that cancer cells notrecognized by the CAR-programmed T cells' targeting receptors can formescape variants that elude the programmed T cells. Thus, while thegenetic re-programming of T cells remains a promising therapy,significant improvements are required in their use to treat solidtumors.

US 2016/0008399 describes an implantable scaffold that greatly improvesthe ability of genetically-reprogrammed lymphocytes (e.g., T cells) totreat solid tumors. The implantable scaffolds includegenetically-reprogrammed lymphocytes, at least one lymphocyte-adhesionmoiety associated with the scaffold; at least one lymphocyte-activatingmoiety associated with the scaffold, and optionally an immune stimulant.Because the scaffold can be implanted at the site of a solid tumor,issues regarding the inefficient homing of the lymphocytes to the tumorsite is overcome.

US 2016/0008399 also describes how the implantable scaffolds createextended second wave protection against tumor cells. Particularly,lymphocytes seeded within the scaffold exit the scaffold followingimplantation and disperse at high densities throughout, for example, atumor resection bed and into draining lymph nodes to destroy remainingresidual tumor cells following a resection. This step releases largeamounts of tumor antigens from dying tumor cells into the tissue. Thetumor antigens are subsequently taken up by antigen presenting cells(APCs). By releasing an immune stimulant, the scaffolds can activateAPCs and tumor-reactive immune cells to mount a robust host anti-tumorimmune response. This “second wave” of anti-tumor immunity is broaderand involves multiple cell types acting in synergy to eliminateremaining tumor cells.

STING (STimulator of INterferon Genes) pathway agonists are a type ofpotent immune stimulant that have been the focus of intense study incancer immunotherapy. However, like previously described adjuvantcompounds (e.g., R848 and related imidazoquinoline TLR7/8 agonists,muramyl dipeptides that trigger (NOD)-like receptors, and RNAoligonucleotide ligands of RIG-I), treatments with unformulated STINGagonists are accompanied by systemic inflammatory toxicity, whichrepresents a major hurdle for using these compounds to treat cancerpatients. Thus, to be clinically effective, high dosages of STINGagonists are required to be repeatedly injected directly into tumorlesions, which limits this therapy to sites that are accessible fordaily inoculations (e.g., skin malignancies).

SUMMARY OF THE DISCLOSURE

The current disclosure continues to advance cancer immunotherapy byproviding improved implantable scaffold. The implantable scaffoldsdisclosed herein are seeded with (i) genetically-reprogrammedlymphocytes; and (ii) at least one lymphocyte-activating moiety. Thus,in particular embodiments, these implantable scaffolds can be simpler inform than those described in US 2016/0008399. In particular embodiments,the implantable scaffolds disclosed herein can also include a STINGagonist. These embodiments and provide a mechanism to harness the potentimmunostimulatory effects of STING agonists, without systemicinflammatory toxicity and without the need for daily injections.

The robustness of the anti-cancer effects observed with the disclosedimplantable scaffolds were synergistic, unexpected, and remarkable. Inalmost half of all subjects tested, complete eradication of solid tumorswas achieved. Thus, tumor cells recognized by thegenetically-reprogrammed lymphocytes as well as tumor cells thatnormally would have become escape variants were effectively treated. Inaddition, following complete eradication of solid tumors, subjects werere-challenged with cancer cells. One hundred percent of re-challengedsubjects failed to develop any measurable tumors demonstrating theeffectiveness of the self-vaccine site. Thus, the implantable scaffoldsdisclosed herein provide a significant advance in the ongoing fightagainst solid tumor cancers.

In particular embodiments, the scaffolds include a scaffold matrixmaterial selected from a micropatterned metallic thin film or a polymer.In particular embodiments, the genetically-reprogrammed lymphocytes areT cells or natural killer (NK) cells. In particular embodiments, the atleast one lymphocyte-activating moiety is selected from interleukin 15and/or antibodies specific for CD3, CD28, and/or CD137. In particularembodiments, the scaffolds include a STING agonist. In particularembodiments, the STING agonist is c-diGMP. In particular embodiments,the STING agonist is embedded within a drug eluting polymer. Inparticular embodiments, the scaffolds include a lymphocyte-adhesionmoiety. In particular embodiments, the lymphocyte-adhesion moiety isselected from fibrin or a GFOGER (SEQ ID NO: 1) peptide.

In particular embodiments, the scaffolds include: (i)genetically-reprogrammed lymphocytes disposed within a Thin Film Nitinol(TFN) micromesh. The genetically-reprogrammed lymphocytes can include Tcells with lymphocyte-activating moieties including antibodies specificfor CD3, CD28, and/or CD137 and/or can include NK cells withlymphocyte-activating moieties including interleukin-15 and antibodiesspecific for CD137. In particular embodiments, these embodiments caninclude the lymphocyte-adhesion moiety fibrin. In particularembodiments, these embodiments can include c-diGMP. In particularembodiments, these embodiments can include c-diGMP embedded within adrug eluting polymer coated onto the surface of the TFN micromesh. Inparticular embodiments, these embodiments can include a STING agonist(e.g., c-diGMP) embedded within particles. If particles are used, one ormore lymphocyte activating moieties may be on the surface of theparticles.

In particular embodiments, a scaffold including a TFN micromesh caninclude a high-density of cells (at least 7×10⁶ per cm² or at least8×10⁶ cells per cm²). TFN micromesh can allow for high cell densities,for example, by packing three layers of cells into each layer of TFNmicromesh.

In particular embodiments, a scaffold including a TFN micromesh can beused as a long-acting scaffold that may continue to deliver lymphocytesfor several days, for more than one week, and/or for more than twoweeks.

In particular embodiments, the scaffolds include (i)genetically-reprogrammed lymphocytes disposed within an alginatescaffold matrix. The genetically-reprogrammed lymphocytes can include Tcells with lymphocyte-activating moieties including antibodies specificfor CD3, CD28, and/or CD137 and/or can include NK cells withlymphocyte-activating moieties including interleukin-15 and antibodiesspecific for CD137. In particular embodiments, these embodiments caninclude a GFOGER (SEQ ID NO: 1) peptide lymphocyte-adhesion moiety. Inparticular embodiments, these embodiments can include c-diGMP. Inparticular embodiments, these embodiments can include c-diGMP embeddedwithin a drug eluting polymer coated onto the surface of the alginatescaffold. In particular embodiments, these embodiments can include aSTING agonist (e.g., c-diGMP) embedded within particles. If particlesare used, one or more lymphocyte activating moieties may be on thesurface of the particles.

BRIEF DESCRIPTION OF THE FIGURES

Many of the drawings submitted herein are better understood in color.Applicants consider the color versions of the drawings as part of theoriginal submission and reserve the right to present color images of thedrawings in later proceedings.

FIG. 1. Solid tumors are heterogeneous and express various levels ofantigens commonly used as targets for therapy. This is a representativeconfocal image of a human pancreatic ductal adenocarcinoma that exhibitsthe substantial diversity of protein expression by these tumors.Cytokeratin expression commonly occurs in adenocarcinomas, so apan-cytokeratin antibody was used to define tumor cell populations. Thetumor differentiation antigen mesothelin is a likely candidate forimmunotherapy, and treatments targeting the cancer stem cell markerEpCAM are currently in clinical development. Scale bar, 100 μm. In colorreproductions of this FIG., pan-cytokeratin antibody is green,mesothelin is red, and EpCAM is blue.

FIGS. 2A-2I. Systemic infusions of tumor-specific CAR-T cells produceonly modest therapeutic benefits. (FIG. 2A) Orthotopic mouse model ofpancreatic ductal adenocarcinoma. (FIG. 2B) Bright field microscopy ofKPC pancreas 7 days after surgical implantation. Hematoxylin/eosinstaining reveals invasive adenocarcinoma interspersed with parenchymaltissue in the head of the pancreas; the adjacent healthy tissueaccentuates the disorganized nature of the neoplastic regions. Theformation of both differentiated and undifferentiated acinar structures(i.e., glands and ducts), which are hallmarks of adenocarcinoma, is alsoclear in these samples. Analysis of magnified (20×) images revealedsubstantial recruitment of immune cells, including polymorphonuclearleukocytes, within the tumor microenvironment. The dashed rectanglelocalizes the magnified insert. Scale bar, 100 μm. (FIG. 2C) Heat maprepresentation of flow cytometry data that quantifies the cell-to-cellvariability in Rae-1 expression by KPC tumors. Seven-day-old tumors(tagged with GFP to distinguish them) were disaggregated intosingle-cell suspensions and labeled with antibodies against Rae-1 somean fluorescent intensities of this antigen could be measured by flowcytometry; the shading indicates relative expression levels compared toan isotype control. Results from 1,800 randomly-chosen cells are shown.(FIG. 2D) This schematic depicts how the chimeric receptor used torecognize Rae-1 includes full-length mouse NGK2D fused to a murine CD3intracellular signaling molecule. (FIG. 2E) Flow cytometry measuringsurface expression of the NKG2D CAR on mouse effector T cells afterretroviral transduction and three days of expansion in medium containingG418. (FIG. 2F) ⁵¹Cr release cytotoxicity assay of NKG2D CAR-transducedT cells reacting with KPC pancreatic tumor cells. (FIG. 2G) Ten daysafter firefly luciferase-expressing KPC tumor cells were transplantedinto the pancreas of albino C57BL/6 mice, the animals were injected with10⁷ NKG2D CAR-transduced T cells. To measure the dynamics of programmedlymphocyte targeting, in parallel experiments mice were injected withCAR-T cells that co-express the click beetle red luciferase reporter.Shown is sequential bioluminescence imaging of the KPC tumors and theadoptively transferred T cells in five representative mice from eachcohort (n=10). (FIG. 2H) Kaplan-Meier survival curves for treated andcontrol mice. Shown are ten mice per treatment group pooled from threeindependent experiments; ms, median survival. Statistical analysisbetween the treated experimental and the untreated control group,depicted here, was done using the Log-rank test and P<0.05 wasconsidered significant. (FIG. 2I) Flow cytometry quantification of Rae-1antigen expression on KPC tumor cells following NKG2D CAR-T celltherapy. Shown are 1,800 randomly-chosen cells.

FIGS. 3A, 3B. Biomatrices placed directly on pancreatic tumors canfunction as an effective delivery platform for CAR-programmed T cells.(FIG. 3A) Brightfield microscopy of stimulatory microspheresincorporated into the scaffold; below is a depiction of microparticlecomposition. Scale bar, 70 μm. (FIG. 3B) This series illustrates adisclosed methodology: [1] Scaffold; [2] Seeding of tumor-reactive Tcells into the device; [3] Incision; [4] Orthotopic KPC pancreatictumor; [5]-[7] Implantation of a T cell-loaded device; [8] Woundclosure; [9] Sustained release of tumor-reactive T cells.

FIGS. 4A-4E. Polymer-launched CAR-T cells robustly expand at the tumorsite and trigger tumor regression, but do not affect cells missing thetarget antigen. (FIG. 4A) Bioluminescence imaging of KPC tumors andadoptively transferred CAR-T cells. Mice were treated with 10⁷NKG2D-transduced lymphocytes injected locally into the tumor, orcontained in bioactive scaffolds implanted directly onto the tumorsurface. Five representative mice from each cohort (n=10) are shown.(FIG. 4B) T cell signal intensities from sequential bioluminescenceimages captured every two days after cell transfer. Each line representsone animal and each dot indicates the whole animal photon count. At theindicated time points, pairwise differences in photon counts betweentreatment groups were analyzed with the Wilcoxon rank-sum test. (FIG.4C) Quantified KPC bioluminescent tumor signal. (FIG. 4D) Kaplan-Meiersurvival curves for treated and control mice. Shown are ten mice pertreatment group pooled from three independent experiments. ms, mediansurvival. Statistical analysis between the treated experimental and theuntreated control group, depicted here, was done using the Log-rank testand P<0.05 was considered significant. (FIG. 4E) Flow cytometryquantification of Rae-1 antigen expression on KPC tumor cells followingNKG2D CAR-T cell therapy. Shown are 1,800 randomly-chosen cells.

FIGS. 5A, 5B. Design of a biomaterial carrier that co-deliversCAR-expressing T cells and an immune stimulant (shown as a vaccineadjuvant in this example) to simultaneously clear heterogeneous cancercells and establish systemic anti-tumor immunity. (FIG. 5A) Schematicdiagrams of a scaffold loaded with CAR-T cells interacting with thetumor bed: Panels 1 and 2 show how factor-containing microspheresincorporated into the device stimulate the expansion of CAR-expressing Tcells and promote their egress into surrounding tissue. APC, antigenpresenting cell. Panels 2 and 3 illustrate the release of vaccineadjuvant from T cell-loaded scaffolds, priming host immune cells torecognize and lyse tumor cells and thereby protect against antigenescape variants. (FIG. 5B) Macro- and microscopic views of a porousalginate matrix functionalized with microparticles with the STINGagonist cyclic-di-GMP (i.e., c-diGMP or cdGMP, which are usedinterchangeably) entrapped in the polymer core and stimulatoryanti-CD3/CD28/CD137 antibodies tethered to its phospholipid membrane.The chemical structure of c-diGMP is shown below.

FIGS. 6A, 6B. Scaffold-released CAR-T cells and STING agonist synergizeto activate host antigen-presenting cells. (FIGS. 6A, 6B) Ten days aftertransplanting luciferase-expressing KPC tumor cells into the pancreas ofmice, scaffolds containing either 7×10⁶ tumor-reactive CAR-T cells, 6 μgc-diGMP, or a combination of both were implanted on the tumor surface;control mice received no treatment. Five days later, peripancreaticlymph nodes were digested into cell suspensions for analysis by flowcytometry. Only lymph nodes that were not engulfed by tumors were used,and they were pooled from at least five animals. (FIG. 6A) Flowcytometry of myeloid maturation markers (CD11 b and CD11c): histogramsshown on the right depict the expression of the costimulatory factorCD86 and MHC class II molecules after gating on CD11c+CD11b+double-positive cell populations. (FIG. 6B) Absolute numbers of matureand activated (CD11b+CD11c+CD86+MHC-II+) dendritic cells inperipancreatic lymph nodes. Points represent the cell number per lymphnode in samples pooled from 5 mice, and the data are representative offour separate studies.

FIGS. 7A, 7B. Co-release of c-diGMP along with CAR-expressing T cellsfrom scaffolds primes endogenous tumor-reactive lymphocytes. (FIGS. 7A,7B) KPC tumors expressing glycoprotein 33 were transplanted into mice.These mice were treated with biomaterial-delivered c-diGMP, CAR-T cells,or a combination of the two and host gp33-specific T cells in theperipheral blood were quantified by tetramer staining. To differentiateendogenous from adoptively transferred T cells, congenic CD45.1recipient mice were used for these studies. (FIG. 7A) Representativeflow cytometry plots showing percentages of gp33 tetramer-positive cellsin peripheral blood 10 days after scaffold implantation, gated onCD45.1+(host) CD8+ cells. Shown profiles are representative of threeindependent experiments. (FIG. 7B) Absolute numbers of primed(CD45.1+CD8+gp33+) T cells. Shown are ten mice pooled from threeindependent experiments. Each bar represents the mean absolute cellcount ±s.e.m. The unpaired Student's t-test was used to test thedifference between absolute cell counts.

FIGS. 8A-8C. Scaffolds that co-deliver STING agonists along withCAR-expressing T cells can limit tumor immune escape. (FIG. 8A) Serialin vivo bioluminescence imaging of KPC-luc tumors. Shown are fiverepresentative mice from each cohort (n=10). (FIG. 8B) Quantified KPCbioluminescent tumor signals; shown are ten mice per treatment grouppooled from three independent experiments. (FIG. 8C) Kaplan-Meiersurvival curves for treated and control mice. ms, median survival.Statistical analysis between the treated experimental and the untreatedcontrol group, depicted here, was done using the Log-rank test andP<0.05 was considered significant.

FIGS. 9A, 9B. Scaffolds can elicit global antitumor immunity. (FIG. 9A)Serial in vivo bioluminescence imaging of KPC-luc tumor cells injectedintravenously into the four mice that experienced complete tumorregression as described in relation to FIGS. 8A-8C. Age-matched naivemice were used as control. (FIG. 9B) Kaplan-Meier survival curves.

FIGS. 10A-10D. (FIG. 10A) Schematic illustrating scaffoldsfunctionalized with lymphocyte-activating moieties (e.g.,anti-CD3/CD28/CD137 antibodies) by covalent coupling of the moieties toa cell scaffold (e.g. Thin Film Nitinol (TFN) micromesh). (FIG. 10B)Schematic illustrating cell scaffolds functionalized withlymphocyte-activating moieties (e.g., anti-CD3/CD28/CD137 antibodies) bycovalent coupling of the moieties to lymphocyte-adhesion moieties (e.g.,fibrin, collagen) covalently coupled to or coated on a cell scaffold(e.g., TFN micromesh). (FIG. 10C) Schematic illustratingthree-dimensional implants formed with multi-layered thin film micromesh(e.g., TFN micromesh) and embedded with an ultra-high density of tumorreactive immune cells (e.g., CAR T cells). (FIG. 10D) Functionalizedmicropatterned metallic thin film (e.g. TFN micromesh) achievesultra-high cell densities.

FIGS. 11A-11H. TFN micromeshes functionalized with appropriatelymphocyte-adhesion moieties and stimulatory cues support rapidmigration and robust expansion of T cells. (FIG. 11A) Photograph of aTFN micromesh. Scale bar: 2 mm. (FIG. 11B) Light microscopy image of anuncoated (left panel) and fibrin coated (right panel) TFN micromesh.Magnification: 40×. Scale bar: 120 μm (FIG. 11C) Electron microscopyimage of an uncoated (left panel) and fibrin coated (right panel) TFNmicromesh. Magnification: 270×1,100× magnified versions are shown inpanel (FIG. 11D). (FIG. 11E) Time-lapse video projections of lymphocytemigration through uncoated (left) and fibrin-coated (right) TFNmicromeshes tracked for 30 min; each shaded color represents anindividual T cell. Scale bar: 50 μm. Shown below are comparison ofaverage velocities and mean T cell displacements, based on 30 randomlychosen cells from two independent experiments. (FIG. 11F) Highmagnification confocal image of human T cells (Alexa 488-labeled: green)entrapped in a fibrin-coated TFN micromesh. Scale bar: 100 μm. The insetshows a magnification. Scale bar: 50 μm. (FIG. 11G) Schematic diagram ofa TFN micromesh functionalized with a T-cell adhesion ligand (fibrin)and stimulatory ligands (anti-CD3/CD28/CD137 antibodies covalentlycoupled to fibrin by EDC chemistry). (FIG. 11H) Representativecarboxyfluorescein succinimidyl ester (CFSE) assay of T cells that haveexited TFN micromeshes during the 7 d test period, in whichproliferation was assessed by measuring CFSE dilution (consequent tocell division) using flow cytometry. Mean CFSE fluorescence intensities(MFI) for the lymphocyte populations are indicated at the upper left.

FIGS. 12A-12I. Sustained release of T cells from bioactive TFNmicromeshes. (12A, 12B) Schematics of a single-layer TFN micromesh (FIG.12A) or stent (FIG. 12B) functionalized with anti-CD3/CD28/CD137antibody-functionalized fibrin and tumor-reactive CAR T cells. (FIG.12C, FIG. 12D). Photomicrographs of a micromesh film or stent. (FIG.12E, FIG. 12F) The egress of lymphocytes from TFN micromesh was measuredby abutting them to a three-dimensional collagen gel (PureCol)containing 10 ng/mL inflammatory cytokine IP-10 and culturing incomplete RPMI medium. Microscopy of a TFN micromesh (FIG. 12E) or astent (FIG. 12F) containing embedded CAR T cells at day 0 or day 2.(FIG. 12G, FIG. 12H) Absolute counts of viable T cells transited fromthese TFN micromeshes (FIG. 12G) or stents (FIG. 12H) into surroundingcollagen matrix. Each line represents one TFN micromesh. Data arerepresentative of three independent experiments. (FIG. 12I) Lymphocytesdemonstrate high persistence on TFN micromesh after 6 days. TFNmicromesh was loaded with CAR-T cells and placed against a tissuemimetic for 6 days. Ultra-high cell densities were still present on thefilm after 6 days, indicating favorable lymphocyte persistence can beobtained at tumor sites.

FIGS. 13A-13B. Launching ovarian cancer-specific CAR T cells frombioactive TFN micromeshes eradicates established multifocal disease. Onemillion OVCAR-3 human ovarian cancer cells (expressing the tumor antigentyrosine kinase-like orphan receptor ROR1 and firefly luciferase) weresurgically implanted into the diaphragm of NOD scid gamma (NSG) mice andallowed to establish for 8 weeks. At that time point, all animalsdeveloped ovarian cancer lesions mimicking spread to the diaphragm inwomen with ovarian cancer. Mice were treated with 10×10⁶ human CAR Tcells specific for ROR-1 injected intravenously, injected locally intotumor lesions or 2×10⁶ cells delivered from an implanted TFN micromesh.(FIG. 13A) Implementation of the approach: [1] Established ovariancancer lesions in the diaphragm; Li: Liver. Diaph: Diaphragm. Tu: Tumor.[2] implantation of the TFN micromesh loaded with anti-ROR1 CAR T cellsbetween the liver and the diaphragm. [3] TFN micromesh afterimplantation. (FIG. 13B) Serial in vivo bioluminescence imaging ofOVCAR-3-luc tumors.

FIG. 14. Kaplan-Meier survival curve for mice treated with the TFNmicromesh lymphocyte scaffold.

FIG. 15. Small molecules (e.g., STING agonists and/or immune stimulants)may be incorporated into lymphocyte scaffolds (e.g., TFN micromeshscaffolds) using a drug eluting polymer.

FIG. 16. Polypeptide sequence of the GFOGER (SEQ ID NO: 1) adhesionmotif.

FIG. 17. Polypeptide sequence of an exemplary GFOGER (SEQ ID NO: 1)peptide (SEQ ID NO: 2).

FIG. 18. Polypeptide sequence of the ICAM-1 cell adhesion molecule (SEQID NO: 3).

FIG. 19. Polypeptide sequence of the FN-III₇₋₁₀ fragment (SEQ ID NO: 4).

FIG. 20. Exemplary sequences that can be used to engineer chimericantigen receptors (SEQ ID NOs: 14-27).

DETAILED DESCRIPTION

Cancer immunotherapy describes a field in which a patient's immunesystem is used to treat cancer. For example, cancer immunotherapyincludes the use of vaccines to immunize patients against thedevelopment of cancer. Unfortunately, the responses cancer vaccines canelicit can require months to mature and are usually insufficient tocontrol advanced disease.

Attempts have also been made to more quickly or potently stimulate apatient's own T cells to attack cancer cells. The repertoire ofreceptors normally expressed by T cells generally has a low affinity toself/tumor antigens, however, so this approach has not achievedsufficient success in the fight against cancer.

An emerging immunotherapy approach involves the alteration ofpatient-derived lymphocytes (e.g., T cells) with genes encoding chimericantigen receptors (CARs) that are engineered to have high affinity forselected macromolecular targets in the tumors. The introduced genes canalso produce costimulatory signals to elicit robust T cell expansion.The method involves retrieval of T cells from a patient and redirectingthem ex vivo to express CARs composed of a tumor-specific single-chainantibody (scFv) fused to costimulatory and CD3 signaling domains, whichenable the programmed cells to lyse tumor targets in a human leukocyteantigen (HLA)-independent fashion.

Therapies that employ such CAR-programmed T cells have consistentlyproduced positive results in patients with hematologic malignancies inclinical trials. However, when it comes to solid tumors, theeffectiveness of these therapies has been limited by: 1) inefficienthoming of the lymphocytes to the tumor site; and 2) theimmunosuppressive microenvironment solid tumors create. Moreover, 3)CAR-programmed T cells only recognize the tumor antigen for which theyhave been programmed. The phenotypic diversity of solid tumors, however,means that cancer cells not recognized by the CAR-programmed T cells canform escape variants that elude the programmed T cells. Thus, while thegenetic re-programming of T cells remains a promising therapy,significant improvements are required in their use to treat solidtumors.

US 2016/0008399 describes an implantable scaffold that greatly improvesthe ability of genetically-reprogrammed lymphocytes (e.g., T cells) totreat solid tumors. The implantable scaffolds includegenetically-reprogrammed lymphocytes, at least one lymphocyte-adhesionmoiety associated with the scaffold; at least one lymphocyte-activatingmoiety associated with the scaffold, and optionally an immune stimulant.

US 2016/0008399 also describes how the implantable scaffolds createextended second wave protection against tumor cells. Particularly,lymphocytes seeded within the scaffold exit the scaffold followingimplantation and disperse at high densities throughout, for example, atumor resection bed and into draining lymph nodes to destroy remainingresidual tumor cells following a resection. This step releases largeamounts of tumor antigens from dying tumor cells into the tissue. Thetumor antigens are subsequently taken up by antigen presenting cells(APCs). By releasing an immune stimulant, the scaffolds can activateAPCs and tumor-reactive immune cells to mount a robust host anti-tumorimmune response. This “second wave” of anti-tumor immunity is broaderand involves multiple cell types acting in synergy to eliminateremaining tumor cells.

STING (STimulator of INterferon Genes) pathway agonists are a type ofpotent immune stimulant that have been the focus of intense study incancer immunotherapy. However, like previously described adjuvantcompounds (e.g., R848 and related imidazoquinoline TLR7/8 agonists,muramyl dipeptides that trigger (NOD)-like receptors, and RNAoligonucleotide ligands of RIG-I), treatments with unformulated STINGagonists are accompanied by systemic inflammatory toxicity, whichrepresents a major hurdle for using these compounds to treat cancerpatients. Thus, to be clinically effective, high dosages of STINGagonists are required to be repeatedly injected directly into tumorlesions, which limits this therapy to sites that are accessible fordaily inoculations (e.g., skin malignancies).

The current disclosure continues to advance cancer immunotherapy byproviding an implantable scaffold seeded with (i)genetically-reprogrammed lymphocytes; and (ii) at least one lymphocyteactivating moiety. This scaffold overcomes the three problems notedabove with cell therapy in solid tumors because 1) the scaffold can beimplanted at the site of a solid tumor, thereby overcoming inefficienthoming of the lymphocytes to the tumor; the immunosuppressive tumormicroenvironment is addressed by the inclusion of lymphocyte stimulatingantibodies in the scaffold which encourage T-cell proliferation andtumor cell killing; and 3) inclusion of STING agonists in such ascaffold provides a mechanism to harness their potent immunostimulatoryeffects without systemic inflammatory toxicity and without the need fordaily injections.

The robustness of the anti-cancer effects observed with the disclosedimplantable scaffolds were synergistic, unexpected, and remarkable. Inalmost half of all subjects tested, complete eradication of solid tumorswas achieved. Thus, tumor cells recognized by thegenetically-reprogrammed lymphocytes as well as tumor cells thatnormally would have become escape variants were effectively treated. Inaddition, following complete eradication of solid tumors, subjects werere-challenged with cancer cells. One hundred percent of re-challengedsubjects failed to develop any measurable tumors demonstrating theeffectiveness of the self-vaccine site. Thus, the implantable scaffoldsdisclosed herein provide a significant advance in the ongoing fightagainst solid tumor cancers.

In particular embodiments, the implantable scaffolds disclosed hereincan also be simpler in form and easier to manufacture than thosedescribed in US 2016/0008399. The implantable scaffolds disclosed hereincan also include a STING agonist

For clarity, within the current disclosure, heterogenous solid tumorsare those including tumor cells that express an antigen targeted by agenetically-reprogrammed lymphocyte (e.g., a genetically-reprogrammedCAR-T cells) and tumor cells that do not express the targeted antigen.Those tumor cells expressing the targeted antigen are targeted solidtumor cells. Tumor cells not expressing the targeted antigen are escapevariants.

Various components of the disclosed implantable scaffolds are nowdescribed in more detail.

Implantable Scaffold Matrix Materials. The structures of implantablescaffolds disclosed herein can be constructed from a variety ofmaterials.

In particular embodiments, the scaffold matrix material includes amicropatterned metallic thin film. Micropatterned metallic thin filmsare formulated out of at least one metallic material, include arepetitive pattern in their structure, and have a thickness of 100 μm orless. Exemplary metals that can be used for a micropatterned metallicthin film include ELGILOY® (Elgiloy Specialty Metals, Elgin, Ill.),stainless steel, and nitinol. In particular embodiments, Thin FilmNitinol (TFN) micromesh is used as a scaffold matrix material (e.g.,Rigberg et al., J Vasc Surg. 2009 August; 50(2):375-80). Nitinol refersto a family of alloys of nickel and titanium that include between 50%and 60% nickel and between 40% and 50% titanium. Up to 2% of the nickelin a nitinol alloy can be replaced with cobalt. TFN micromesh is avariant of the bulk material that is produced on micropatterned siliconwafers via a sputter deposition process. A particular advantage of TFNmicromesh is that because it is based on photolithographic technology,one can exert exquisite control over the size and shape of the TFNmicromesh pores. TFN micromesh can refer to nitinol in the shape of athin sheet or film (e.g., 100 μm, 10 μm, 1 μm, or 0.1 μm thick). Toenhance the cell cargo capacity of single-layer TFN micromeshes,sandwich-like layer-by-layer thin films with alternating TFN- andcells-layers can be fabricated into three-dimensional structures (asillustrated in FIG. 100).

In particular embodiments, the scaffold matrix material includesbiocompatible polymers. Exemplary biocompatible polymers include agar,agarose, alginate, alginate/calcium phosphate cement (CPC),beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose),cellulose, chitin, chitosan (see, for example, Levengood et al., J.Mater. Chem. B, 2014, 2, 3161-3184 describing porous chitosanscaffolds), collagen, elastin, gelatin, hyaluronic acid collagen,hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx),poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide)(PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA;see, for example, Omar et al., Sci. Transl. Med. 2009 Nov. 25; 1(8):8ra19 describing porous PLGA scaffolds), polypropylene oxide (PPO),poly(vinyl alcohol) (PVA), silk, soy protein, and soy protein isolate,alone or in combination with any other polymer composition, in anyconcentration and in any ratio. Blending different polymer types indifferent ratios using various grades can result in characteristics thatborrow from each of the contributing polymers. Various terminal groupchemistries can also be adopted.

When injectable implantable scaffolds are used, the scaffold matrix(e.g., polymers) can be responsive to a changed environmental conditionfollowing implantation. Polymers with these characteristics are known tothose of ordinary skill in the art. For example, in particularembodiments, an injectable in situ gel-forming system is used. Inparticular embodiments, the polymer formulation can gel in vivo inresponse to temperature change (thermal gelation), in response to pHchange or in response to light. For example, polymers that gel inresponse to ultraviolet (UV) light can be used. In particularembodiments, the polymer formulation can gel in vivo in response toionic cross-linking. In particular embodiments, the polymer formulationcan gel in vivo in response to solvent exchange. In particularembodiments, the gel used is thermoreversible, pH reversible, or lightreversible. In particular embodiments, the gel used is high-viscosityand shear-thinning. In additional gelling embodiments, the gel can be agel formed from any appropriate polymer. Injectable, spontaneouslyassembling scaffolds fabricated from mesoporous silica rods aredescribed in Kim et al., Nature Biotechnology 33, 64-72 (2015).

Self-assembling peptide scaffolds can also be used as scaffold matrixmaterials (see, e.g., Zacco et al., Biomacromolecules. 2015 Jul. 13;16(7): 2188-97).

In particular embodiments, alginate is used as a scaffold matrixmaterial, either separately or in combination with one or more othermaterials. Alginate is easily processed, water soluble, andnon-immunogenic. Alginate is a biodegradable anionic polysaccharide withfree hydroxyl groups that offer easy gelling. In alternativeembodiments, the polymer may be a polyelectrolyte complex mixture (PEC)formed from a 1:1 solution of alginate and chitosan.

In particular embodiments, a structure (e.g., scaffold matrix material)may formed from an alginate/calcium carbonate/glucono-delta-lactonemixture, such as 0.5-5% alginate, 0.5-15 g/L calcium carbonate, and 1-50g/L gluconon-delta-lactone in a ratio of 2:1:1 (alginate:CaCO₃:GDL).Polymer structures may also include varying amounts of gelatin incombination with varying amounts of alginate. Depending on the materialsand material ratios in mixture, the structures may optionally becross-linked. Collagen/alginate hybrid scaffolds such as those describedin Lee at al., Chem. Mater., 2012, 24(5), 881-891 can also be used.

In particular embodiments, polymer solutions having varying amounts ofpolymer dissolved in an acidic solution can be used to form thestructures disclosed herein. The concentration of the acid can beadjusted depending on the amount of polymer dissolved. In one aspect,the acidic solution is 1% (v/v) acetic acid. In particular embodiments,the amount of polymer in solution is between 0.5-5% (w/v) and any wholeor partial increments therebetween. For example, the amount of polymerin solution (w/v) can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or5%. In particular embodiments, the amount of polymer in solution is 2.4%(w/v). In other various embodiments, the polymer is dissolved in atleast one of water, acid, acetic acid, camphene, orcamphene-naphthalene.

When gelatin is incorporated, the concentration of the acid can beadjusted depending on the amount of gelatin in combination with polymer(in particular embodiments, alginate) that is dissolved. In one aspect,the acidic solution is 1% (v/v) acetic acid. In particular embodiments,the amount of gelatin in solution is between 1-10% (w/v) and any wholeor partial increments therebetween. For example, the amount of alginatein solution (w/v) can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. Inparticular embodiments, the amount of alginate in solution is 5.5%(w/v). In particular embodiments, the polymer solution includes acombination of 2.4% (w/v) alginate solution and a 5.5% (w/v) gelatinsolution. In other various embodiments, the gelatin in combination withvarying amounts of alginate is dissolved in at least one of water, acid,acetic acid, camphene, or camphene-naphthalene.

In particular embodiments, polymer-based scaffolds can be formed asfollows: a weight by volume (w/v) polymer solution in deionized (DI)water can be prepared and filtered with a 0.45 micrometer bottle filterto remove any particles and then frozen to −80° C. The frozen sample canbe lyophilized in a 4.5 liter benchtop freeze dry system (Labconco,Kansas City, Mo.). The filtered lyophilized polymer can be reconstitutedinto solutions of various concentrations (0.1%-5%) with water or buffer.

Crosslinking can be performed with, for example, calcium chloride and/orcalcium carbonate. Calcium carbonate is a slow crosslinker, with samplestaking up to several hours to fully crosslink. To increase the speed ofthe reaction gluconodeltalactone (GDL) can be added. Calcium chloride isa fast crosslinker and the samples will fully gel in a few minutes. Inone method, the addition of CaCl₂ to the polymer solution can occurprior to freezing. Other methods include use of a 5.5% (w/v) solution ofcalcium carbonate+GDL added to the polymer solution prior to initialfreezing.

In particular embodiments, polymer solutions can be degassed in a speedmixer and poured slowly into casts to prevent bubbles from forming. Whenpipetting the polymer solutions into small molds, air bubble formationcan be avoided by placing a micropipette on the open end of mold groovesand repeatedly flushing the entire canal system until the residual airis flushed out.

Freeze casting can be used to form the scaffolds disclosed herein.Various polymer solutions can be freeze cast into various sized casts aswould be understood by those skilled in the art. The rate of coolingshould be controlled as it affects the size and alignment of pores, aswell as the formation of ridges. In particular embodiments, the coolingrate can range between 0.1-100° C. per minute (m) and any whole orpartial increments therebetween. In particular embodiments, the coolingrate can range between 1-10° C./m, and any whole or partial incrementstherebetween. For example, the cooling rate (° C./m) can be 0.1, 0.5, 1,2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

In particular embodiments, in preparing an alginate scaffold, analginate/calcium carbonate/glucono-delta-lactone mixture can be preparedby stirring, with concentrations ranging from 0.5-5 wt % alginate,0.5-15 g/L calcium carbonate, and 1-50 g/L glucono-delta-lactone in avolume ratio of 2:1:1 (alginate:CaCO₃:GDL) as a “pre-gelling” process.In particular embodiments, the resulting mixture can be freeze cast(directionally frozen) at a constant cooling rate (0.1°/min-10°/min)until solid and lyophilized until dry. The dried scaffolds can becrosslinked in 0.1-2.5 wt. % calcium chloride for 5-30 minutes andwashed in HEPES buffered saline prior to any further use of thescaffold.

In particular embodiments, in preparing an alginate-chitosan scaffold,an alginate-chitosan polyelectrolyte complex (PEC) mixture can beprepared by sonicating or homogenizing on ice in a range of 1:1 to 1:9solutions (both ways) of alginate (prepared in water) and chitosan(prepared in 1% acetic acid) and total polymer content ranging from0.5%-5%. The pH of the resulting mixture can be adjusted with NaOH up to10.0. In particular embodiments, the alginate-chitosan PEC mixture canbe freeze cast at a constant cooling rate (0.1°/min-10°/min) until solidand lyophilized until dry. Dried scaffolds can be crosslinked in0.1-2.5% calcium chloride for 5-30 minutes and washed in PBS prior toany further use of the scaffold.

Implantable scaffolds can also be manufactured from various materialsusing 3D bioprinting (see, e.g., Singh et al., Polymers 2016, 8(1), 19;and An et al., Engineering, Volume 1, Issue 2, June 2015, Pages261-268).

Lymphocyte-Activating Moieties. Particular embodiments of theimplantable scaffolds disclosed herein may also includeLymphocyte-Activating Moieites (LAM). LAM include any compound thatactivates a lymphocyte and can be incorporated in or attached to theimplantable scaffolds disclosed herein. Activation of a lymphocyterefers to the state of a lymphocyte that has been sufficientlystimulated to induce detectable cellular proliferation, cytokineproduction, or effector function such as tumor targeting and/or killing.If the lymphocyte is a T-cell, activation also results in expression ofcell surface markers particular to the T-cell type. Exemplary LAMinclude IL-15, CD3, CD27, CD28, CD80, CD86, 4-1BB, CD137, OX40, CD30,CD40, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands orantibodies, CD1d, recombinant CD1d molecules preloaded with α-galactosylceramide and/or recombinant major histocompatibility complex (MHC)molecules loaded with defined tumor antigens or peptides to selectivelyexpand particular lymphocyte types embedded within a scaffold. ExemplaryLAM for NK cells include IL-15 and CD137.

STING Agonists. “STING” is an abbreviation of “stimulator of interferongenes”, which is also known as “endoplasmic reticulum interferonstimulator (ERIS)”, “mediator of IRF3 activation (MITA)”, “MPYS” or“transmembrane protein 173 (TM173)”. STING is a transmembrane receptorprotein and is encoded by the gene TMEM173 in human. Activation of STINGleads to production of Type I interferons (e.g. IFN-a and IFN-β), viathe IRF3 (interferon regulatory factor 3) pathway; and to production ofpro-inflammatory cytokines (e.g. TNF-a and IL-1β), via the NF-κB pathwayand/or the NLRP3 inflammasome.

Human and murine STING are naturally activated two ways: via binding ofexogenous (3 ‘,3) cyclic dinucleotides (c-diGMP, c-diAMP and c-GAMP)that are released by invading bacteria or archaea; and via binding of(2’,3′)cyclic guanosine monophosphate-adenosine monophosphate((2′,3′)c-GAMP), an endogenous cyclic dinucleotide that is produced bythe enzyme cyclic GMP-AMP synthase (cGAS; also known as C6orfl50 orMB21D1) in the presence of exogenous double-stranded DNA (e.g. thatreleased by invading bacteria, viruses or protozoa).

The term “STING agonist” refers to a substance that activates the STINGreceptor in vitro or in vivo. A compound can be deemed a STING agonistif: (i) induces Type I interferons in vitro in human or animal cellsthat contain STING; and (ii) does not induce Type I interferons in vitroin human or animal cells that do not contain STING or does not containfunctioning STING. A typical test to ascertain whether a ligand is aSTING agonist is to incubate the ligand in a wild-type human or animalcell line and in the corresponding cell line in which the STING codinggene has been genetically inactivated by a few bases or a longerdeletion (e.g. a homozygous STING knockout cell line). An agonist ofSTING will induce Type I interferon in the wild-type cells but will notinduce Type I interferon in the cells in which STING is inactivated.

In particular embodiments, STING agonists include cyclic molecules withone or two phosphodiester linkages, and/or one or two phosphorothioatediester linkages, between two nucleotides. This includes (3′,5′)-(3′,5′)nucleotide linkages (abbreviated as (3′,3′)); (3′,5′)-(2′,5′) nucleotidelinkages (abbreviated as (3′,2′)); (2′,5′)-(3′,5′) nucleotide linkages(abbreviated as (2′,3′)); and (2′,5′)-(2′,5′) nucleotide linkages(abbreviated as (2′,2′)). “Nucleotide” refers to any nucleoside linkedto a phosphate group at the 5′, 3′ or 2′ position of the sugar moiety.

In particular embodiments, STING agonists include compounds of theformula:

In particular embodiments, R1 and R2 may be independently 9-purine,9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or9-isoguanine, as shown below:

In particular embodiments, the STING agonist can include dithio-(R_(P),R_(P))-[cyclic[A(2′,5′)pA(3′,5′)p]] (also known as 2′-5′, 3′-5′ mixedphosphodiester linkage (ML) RR-S2 c-di-AMP or ML RR-S2 CDA), MLRR-S2-c-di-GMP (ML-CDG), ML RR-S2 cGAMP, or any mixtures thereof.

The structure of c-diGMP is shown in FIG. 5B. The structure of c-diAMPincludes:

The structure of c-GAMP includes:

Additional particular examples of STING agonists include:

Name Structure c-AIMP

(3′,2′)c-AIMP

(2′,2′)c-AIMP

(2′,3′)c-AIMP

c-AIMP(S)

c-(dAMP-dIMP)

c-(dAMP-2′FdIMP)

c-(2′FdAMP-2′FdIMP)

(2′,3′)c-(AMP-2′FdIMP)

c-[2′FdAMP(S)- 2′FdIMP(S)]

c-[2′FdAMP(S)- 2′FdIMP(S)](POM)²

Examples of STING agonists also include DMXAA:

Additional examples of STING agonists are described in WO2016/145102.

The optional uses of additional immune stimulants andlymphocyte-adhesion moieties are described next.

Additional Immune Stimulants. In particular embodiments, immunestimulants in addition to STING agonists can be included. In particularembodiments, the immune stimulant is a cytokine, an antibody, a smallmolecule, an siRNA, a plasmid DNA, and/or a vaccine adjuvant.

Exemplary cytokines include IL-2, IL-7, IL-12, IL-15, IL-18, IL-21,TNFα, IFN-α, IFN-β, IFN-γ, or GM-CSF. In particular embodiments, theimmune stimulant may be a cytokine and or a combination of cytokines,such as IL-2, IL-12 or IL-15 in combination with IFN-α, IFN-β or IFN-γ,or GM-CSF, or any effective combination thereof, or any other effectivecombination of cytokines. The above-identified cytokines stimulateT_(H)1 responses, but cytokines that stimulate T_(H)2 responses may alsobe used, such as IL-4, IL-10, IL-11, or any effective combinationthereof. Also, combinations of cytokines that stimulate T_(H)1 responsesalong with cytokines that stimulate T_(H)2 responses may be used.

Exemplary antibodies include anti-PD1, anti-PDL1, anti-CTLA-4,anti-TIM3, agonistic anti-CD40, agonistic anti-4-1BB, and/or bispecificantibodies (e.g., BITE-antibodies: anti-CD3/anti-tumor antigen).Exemplary small molecule drugs include, TGF-beta inhibitors,SHP-inhibitors, STAT-3 inhibitors, and/or STAT-5 inhibitors. Any siRNAcapable of down-regulating immune-suppressive signals or oncogenicpathways (such as kras) can be used whereas any plasmid DNA (such asminicircle DNA) encoding immune-stimulatory proteins can be used.Exemplary vaccine adjuvants, include any kind of Toll-like receptorligand or combinations thereof (e.g. CpG, Cpg-28 (a TLR9 agonist),Polyriboinosinic polyribocytidylic acid (Poly(I:C)), α-galactoceramide,MPLA, Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx),IMO-2055 (EMD1201081), TMX-101 (imiquirnod), MGN1703 (a TLR9 agonist),G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A),Entolimod (a derivative of Salmonella flagellin also known as CBLB502),Hiltonol (a TLR3 agonist), and Imiquimod), and/or inhibitors ofheat-shock protein 90 (Hsp90), such as 17-DMAG(17-dimethylaminoethylamino-17-demethoxygeldanamycin).

Immune stimulants derived from the molecules noted in the precedingparagraphs can also be used. For example, RLI is an IL-15-IL-15receptor-α fusion protein that exhibits 50-fold greater potency thanIL-15 alone. IL-15 impacts the anti-tumor immune response at multiplepoints. It can differentiate monocytes into stimulatory antigenpresenting cells; promote the effector functions and proliferation oftumor-reactive T-cells; and recruit and activate NK cells.

Lymphocyte-Adhesion Moieties. The disclosed scaffolds can includelymphocyte-adhesion moieties to promote lymphocyte mobility out of theimplanted scaffolds. Lymphocyte-adhesion moieties include cell-adhesionmoieties such as cell-adhesion polypeptides that mimic the extracellularmatrix (such as collagen). As used herein, “cell adhesion polypeptides”refer to compounds having at least two amino acids per molecule whichare capable of binding via cell surface molecules, such as integrin. Thecell adhesion polypeptides may be any of the proteins of theextracellular matrix which are known to play a role in cell adhesion,including fibrin, fibronectin, vitronectin, laminin, elastin,fibrinogen, collagen types I, II, and V, as described in Boateng et al.,Am. J. Physiol.—Cell Physio. 288:30-38 (2005). Additionally, the celladhesion polypeptides may be any peptide derived from any of theseproteins, including fragments or sequences containing the bindingdomains. Cell adhesion polypeptides include those havingintegrin-binding motifs, such as the ICAM-1 motif, and related peptidesthat are functional equivalents. Cell adhesion polypeptides may also beany of the peptides described in U.S. Patent Publication No.20060067909.

In particular embodiments, the structures include compounds havinglymphocyte-adhesion moieties, such as a ligand for α₁β₁ integrin, aligand for α₂β₁ integrin, a ligand for α₄β₁ integrin, a ligand for α₅β₁integrin, a ligand for lymphocyte function-associated antigen (LFA-1),or combinations thereof. In particular embodiments, the ligand interactsspecifically with one integrin. In particular embodiments, the ligand isnot a complete fibronectin molecule or is not a complete collagenmolecule.

The lymphocyte-adhesion moiety can be a peptide, antibody, or a smallorganic molecule. A small organic molecule refers to a carbon-basedmolecule having a molecular weight of 500 daltons or less. The antibodyor an integrin binding fragment thereof can be single chained,humanized, or chimeric. In particular embodiments, thelymphocyte-adhesion moiety can be a collagen-mimetic peptide, forexample a stable triple-helical, collagen-mimetic peptide that containsthe GFOGER (SEQ ID NO: 1) adhesion motif from type I collagen, GFP*GER(SEQ ID NO: 1), wherein P* is 4-hydroxyproline, which is recognized bythe α₂β₁ integrin. This peptide adopts a stable triple-helicalconformation similar to the native structure of type I collagen. “GFOGER(SEQ ID NO: 1) peptide” can refer to a collagen-mimetic peptidecontaining a GFOGER (SEQ ID NO: 1) adhesion motif. An exemplary GFOGER(SEQ ID NO: 1) peptide sequence is GGYGGGPC(GPP)₅GFP*GER(GPP)₅GPC (SEQID NO: 2), wherein P* is 4-hydroxyproline.

Particular embodiments utilize ICAM-1 as a lymphocyte-adhesion moiety.ICAM-1 is an Ig-like cell adhesion molecule that binds integrinspromoting cell-cell adhesion and is a ligand for lymphocytefunction-associated (LFA) antigens. ICAM-1 is found primarily onmonocytes and endothelial cells, and is widely inducible, orupregulated, on many cells including T-cells, B-cells, thymocytes,dendritic cells, endothelial cells, fibroblasts, keratinocytes,chondrocytes, and epithelial cells. This protein has a co-stimulatoryeffect upon cytotoxic T-cell interaction, and is utilized in a number ofintercellular binding interactions. In particular embodiments, ICAM-1includes SEQ ID NO: 3.

Particular embodiments utilize FNIII₇₋₁₀ as a lymphocyte-adhesionmoiety. FNIII₇₋₁₀ is a fibronectin fragment spanning the 7-10th type IIIrepeats of fibronectin. The sequence of fibronectin is known in the art.In particular embodiments, FNIII₇₋₁₀ includes SEQ ID NO: 4.

Various methods can be utilized to incorporate LAM, STING agonists,additional immune stimulants and/or lymphocyte-activating moieties intoor onto the implantable scaffolds disclosed herein. For purposes of thisdiscussion the LAM, STING agonists, additional immune stimulants and/orlymphocyte-activating moieties are referred to as “components.”

Components can be found within injectable forms of the structures orembedded within the pores of the scaffolds, attached to the surface ofthe scaffolds, coated on to the surface of the scaffolds, and/orembedded within the scaffolds themselves.

Within particular embodiments, components may be incorporated into thebackbone of a polymer chain. For example, a polymer can be createdcontaining YIGSR (SEQ ID NO: 5) in the backbone as described in Jun etal., J. Biomaterials Sci., Polymer Ed. 15(1), 73-94 (2004).

In particular embodiments, the components may be grafted onto a polymer.In one method, polymers having side branches containing reactivefunctional groups such as epoxide, halide, amine, alcohol, sulfonate,azido, anhydride, or carboxylic acid moieties can be covalently linkedto the amine terminus of polypeptides via the reactive side branchesusing conventional coupling techniques such as carbodiimide reactions.For example, RGD (Arg-Gly-Asp)-containing peptides have been graftedonto the backbone of polymers as described in Lin, et al., J. BiomedicalMaterials Res, 28(3), 329-42 (1994). In another example, RGD-containingpeptides have been grafted onto the side branches of polyethylene glycolbased polymers, as described in Hansson, et al., Biomaterials, 26,861-872 (2005). In particular embodiments, components can be directlycoupled to an implantable scaffold backbone (illustrated in FIG. 10A),or coupled to the lymphocyte-adhesion moieties (e.g. Fibrin, Collagen;illustrated in FIG. 10B) using carbodiimide chemistry prior to scaffoldformation (e.g., molding).

The advantage of these approaches from a manufacturing perspective isthat the implanted scaffolds are entirely composed of a singlebiodegradable material without using particles as a second component. Inaddition, the strategy of integrating LAM into the scaffold can bypassthe need to use lymphocyte-adhesion moieties. Without being bound bytheory, in these embodiments, scaffold-embedded lymphocytes migratealong the displayed LAM (e.g., anti-CD3, anti-CD28, anti-CD137antibodies, IL-15), which serve as adhesion molecules and stimulatorycues. This approach renders the use of particles and lymphocyte-adhesionmoieties optional in embodiments disclosed herein.

In manufacturing, 3D bioprinting can better ensure well-defined scaffoldporosity and composition and could facilitate GMP-manufacturing. In thisscenario, components could also be printed into the scaffolds to betterensure controlled spatial distribution of these components within thescaffold.

As previously indicated, TFN micromesh is a variant of the bulk materialthat is produced on micropatterned silicon wafers via a sputterdeposition process. A particular advantage of TFN micromesh is thatbecause it is based on photolithographic technology, one can also exertexquisite control over the size and shape of its micromesh pores.

In particular embodiments, scaffolds can also be coated with a bioactivecoating (e.g., a drug eluting polymer) including one or more components.In particular embodiments, the scaffold is at least partially coatedwith a bioactive coating. The bioactive coating can be applied onto thesurface of the scaffold in various ways, including the use of coatingmethods that are known in the art. For example, the bioactive coating(e.g., a drug eluting polymer) may be sprayed onto the scaffold by aconventional electrostatic spraying process, resulting in chargeddroplets being deposited onto the surface of the scaffold. As thecoating fluid dries, the components, for example, polypeptides and/orsmall molecules, remain adhered to the surface of the scaffold, forexample, by inter-molecular bonding with the side-chain groups on thepolypeptides. In particular embodiments, the deposited bioactive coatingmay form a monolayer on the surface of the scaffolding.

In particular embodiments, the bioactive coating may be bonded to thesurface of a scaffold by any type of chemical or physical bonding means,including covalent, polar, ionic, coordinate, metallic, electrostatic,or intermolecular dipolar (including Van der Waals) bonds. Bioactivecoatings can additionally include other components to alter the surfaceof the scaffold, for example polylysine, polyornitine, or otherglycoproteins.

Exemplary biocompatible polymers that can be used as drug elutingpolymers include agar, agarose, alginate, alginate/calcium phosphatecement (CPC), beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyla-D-galactose), cellulose, chitin, chitosan (see, for example, Levengoodet al., J. Mater. Chem. B, 2014, 2, 3161-3184 describing porous chitosanscaffolds), collagen, elastin, gelatin, hyaluronic acid collagen,hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx),poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide)(PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA;see, for example, Omar et al., Sci. Transl. Med. 2009 Nov. 25; 1(8):8ra19 describing porous PLGA scaffolds), polypropylene oxide (PPO),poly(vinyl alcohol) (PVA), silk, soy protein, and soy protein isolate,alone or in combination with any other polymer composition, in anyconcentration and in any ratio.

In particular embodiments, for placement of a bioactive coating,surfaces of the scaffolds can be coated in polylysine or polyornithine(0.1-1.0 mg/ml for 3-10 minutes) followed by coating in a protein, suchas a LAM (e.g., anti-CD3, anti-CD28, and/or anti-CD137 antibodies,and/or IL-15) and/or a GFOGER (SEQ ID NO: 1) peptide or fibrin (10μg/ml-250 μg/ml for 30 minutes-24 hours).

In particular embodiments, for placement of a bioactive coating,surfaces of the scaffolds can be coated in polylysine or polyornithine(0.5 mg/ml for 6 minutes) followed by coating in a protein, such as aLAM (e.g., anti-CD3, anti-CD28, and/or anti-CD137 antibodies and/orIL-15) and/or a GFOGER (SEQ ID NO: 1) peptide or fibrin (10 μg/ml-250μg/ml for 30 minutes-24 hours).

In particular embodiments, the surface of the scaffold is coated withLAM and/or a GFOGER (SEQ ID NO: 1) peptide. As an example, the purifiedLAM and/or GFOGER (SEQ ID NO: 1) peptide or fibrin could be stored as atrifluoroacetic acid (TFA) salt and reconstituted to 10 mg/mL in 0.1%TFA and 0.01% sodium azide and stored at 4° C. prior to use. After thescaffolds are rinsed with ethanol to remove contaminants, cleaned infresh ethanol, rinsed in ddH₂O, they can be soaked in phosphate bufferedsaline (PBS). The LAM and/or GFOGER (SEQ ID NO: 1) peptide or fibrin canthen be absorbed onto the scaffolds passively by incubating thescaffolds in a solution of LAM and/or GFOGER (SEQ ID NO: 1) peptide orfibrin in PBS. Prior to implantation, scaffolds could be rinsed in PBSto remove unbound peptides.

In particular embodiments, a bioactive coating such as a drug elutingpolymer can be coated on a scaffold matrix material (e.g., TFN micromeshor alginate). A drug eluting polymer may, for example, contain a smallmolecule (e.g., a STING agonist), and may slowly elute the smallmolecule over time (e.g., 3, 4, or 5 days, 1 week, or 2 weeks).

In particular embodiments, a drug eluting polymer (e.g., PLGA) directlycoats the surface of the scaffold matrix as a monolayer, and othercomponents of the lymphocyte scaffold (e.g., lymphocyte-activatingmoieties) may be applied over the drug eluting polymer. A drug elutingpolymer can be useful, for example, for slowly eluting a small moleculeor drug from the coating into an environment where the lymphocytescaffold is implanted.

As indicated, components can be incorporated directly into or onto thestructure of an implantable scaffold. In particular embodiments,components can be incorporated into or onto particles. Porous particlescan be constructed from any material capable of forming a porousnetwork. Exemplary materials include a variety of material such asbiocompatible polymers, metals, transition metals and metalloids.Exemplary biocompatible polymers include agar, agarose, alginate,alginate/calcium phosphate cement (CPC), beta-galactosidase (β-GAL),(1,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan,collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite,poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide),poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG),poly(lactic-co-glycolic acid) (PLGA), poly(vinyl alcohol) (PVA), silk,soy protein, and soy protein isolate, alone or in combination with anyother polymer composition, in any concentration and in any ratio.Blending different polymer types in different ratios using variousgrades can result in characteristics that borrow from each of thecontributing polymers. Various terminal group chemistries can also beadopted. Exemplary metals, transition metals and metalloids includelithium, magnesium, zinc, aluminum and silica. In particularembodiments, the porous particles include silica. The exceptionally highsurface area of mesoporous silica (exceeding 1,000 m2/g) enables STINGagonist loading at levels exceeding conventional carriers such asliposomes or polymer conjugates. In additional embodiments, pores rangein size from 10-20 nm.

Useful particles of particular embodiments also include those based onlipid-based delivery systems, including cationic lipids, ionizablecationic lipids, lipid-like molecules and pH-sensitive amphiphiles;and/or (ii) dendrimers (highly branched, spherical macromoleculessynthesized from poly-amidoamine (PAMAM) and poly-propylene iminie(PPI), and block copolymers such as PAA/BMA/DMAEMA and PDMAEMA.

The particles can be a variety of different shapes, includingspheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and thelike. The components can be included in the porous nanoparticles in avariety of ways. For example, the components can be encapsulated in theporous particles. In other aspects, the components can be associated(e.g., covalently and/or non-covalently) with the surface or closeunderlying vicinity of the surface of the particles. In particularembodiments, the components can be incorporated in the particles e.g.,integrated in the material of the particles. For example, the componentscan be incorporated into a polymer matrix of polymer particles. One ofordinary skill in the art will appreciate the various ways to carry thecomponents within an implantable scaffold as described herein.

In particular embodiments, particles include liposomes. Liposomes aremicroscopic vesicles including at least one concentric lipid bilayer.Vesicle-forming lipids are selected to achieve a specified degree offluidity or rigidity of the final complex. In particular embodiments,liposomes provide a lipid composition that is an outer layer surroundinga particle.

Liposomes can be neutral (cholesterol) or bipolar and includephospholipids, such as phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylinositol (PI), andsphingomyelin (SM) and other type of bipolar lipids includingdioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain lengthin the range of 14-22, and saturated or with one or more double C═Cbonds. Examples of lipids capable of producing a stable liposome, alone,or in combination with other lipid components are phospholipids, such ashydrogenated soy phosphatidylcholine (HSPC), lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebro sides,distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE) anddioleoylphosphatidylethanolamine4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal). Additionalnon-phosphorous containing lipids that can become incorporated intoliposomes include stearylamine, dodecylamine, hexadecylamine, isopropylmyristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetylpalmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylicpolymers, polyethyloxylated fatty acid amides, and the cationic lipidsmentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA,DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidicacid (PA), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are ableto form vesicles. In particular embodiments, lipids used to createliposomes disclosed herein include cholesterol, hydrogenated soyphosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipidPEG-DSPE.

Methods of forming liposomes are described in, for example, U.S. Pat.Nos. 4,229,360; 4,224,179; 4,241,046; 4,737,323; 4,078,052; 4,235,871;4,501,728; and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys.Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986).

The size of the particles can vary over a wide range and can be measuredin different ways. For example, particles of the present disclosure canhave a minimum dimension of 100 nm. The particles of the presentdisclosure can also have a minimum dimension of equal to or less than500 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40nm, less than 30 nm, less than 20 nm, or less than 10 nm. In particularembodiments, the particles can have a minimum dimension ranging between5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm,between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and60 nm. In particular embodiments, the dimension is the diameter of theparticles. In particular embodiments, a population of particles can havea mean minimum dimension of equal to or less than 500 nm, less than 100nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm,less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, orless than 10 nm. In particular embodiments, a population of particles inan implantable scaffold can have a mean diameter ranging between 5 nmand 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm.Dimensions of the particles can be determined using, e.g., conventionaltechniques, such as dynamic light scattering and/or electron microscopy.

In particular embodiments, the scaffolds include protocells asparticles. Protocells can be formed via fusion of liposomes to poroussilica nanoparticles. The high pore volume and surface area of thespherical mesoporous silica core allow high-capacity encapsulation of aspectrum of cargos, including components. The supported lipid bilayer,whose composition can be modified for specific biological applications,can serve as a modular, reconfigurable scaffold, allowing the attachmentof a variety of functional molecules, such as the components describedelsewhere herein.

When desired, release of various materials from particles can bemodified by incorporation of surfactants, detergents, complexing agents,internal phase viscosity enhancers, surface active molecules,co-solvents, chelators, stabilizers, derivatives of cellulose,polysorbates, PVA or sucrose. Salts and buffers can also be used toalter release characteristics.

In particular embodiments a lymphocyte scaffold including a TFNmicromesh can be coated on a medical device. TFN is unique among cellscaffolds in that it can be easily incorporated into aminimally-invasive medical device, such as a stent or anothercylindrical-shaped device. Minimally invasive medical device can referto a medical device that can be placed/implanted using a minimallyinvasive procedure. A minimally invasive procedure can be a procedurethat requires only a very small incision (e.g., less than 1 cm or lessthan 2 cm), and/or is associated with shortened wound healing time,associated pain or risk of infection, as compared to procedures thatrequire larger incisions. An example of a stent covered with a TFNmicromesh is shown in FIG. 12D. This unique device facilitates deliveryof anti-cancer lymphocytes directly to the site of a solid tumor withoutthe need for open surgery.

Genetically Reprogrammed Lymphocytes. The structures of the scaffoldsdisclosed herein include embedded lymphocytes. Any type of lymphocytecapable of targeting and killing tumor cells, targeting tumor cells forkilling by other cell types, or otherwise mediating tumor cell killingcan be used. The lymphocytes can be autologous to the individual to whomthe scaffold is administered.

Lymphocytes include T-cells, B cells and natural killer (NK) cells. Thecurrent disclosure focuses on the use of embedded T-cells and/or NKcells, but other types of lymphocytes may be used as well, alone or incombination.

Several different subsets of T-cells have been discovered, each with adistinct function. T-cells include helper cells (CD4+ T-cells) andcytotoxic T-cells (CTLs, CD8+ T-cells) which include cytolytic T-cells.

T helper cells assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and activation ofcytotoxic T-cells and macrophages, among other functions. These cellsare also known as CD4+ T-cells because they express the CD4 protein ontheir surface. Helper T-cells become activated when they are presentedwith peptide antigens by MHC class II molecules that are expressed onthe surface of antigen presenting cells (APCs). Once activated, theydivide rapidly and secrete small proteins called cytokines that regulateor assist in the active immune response.

Cytotoxic T-cells destroy virally infected cells and tumor cells, andare also implicated in transplant rejection. These cells are also knownas CD8+ T-cells because they express the CD8 glycoprotein at theirsurface. These cells recognize their targets by binding to antigenassociated with MHC class I, which is present on the surface of nearlyevery cell of the body.

A majority of T-cells have a T-cell receptor (TCR) existing as a complexof several proteins. The actual TCR is composed of two separate peptidechains, which are produced from the independent T-cell receptor alphaand beta (TCRα and TCRβ) genes and are called α- and β-TCR chains.Gamma-delta (γΔ) T-cells represent a small subset of T-cells thatpossess a distinct TCR on their surface. However, in γΔ T-cells, the TCRis made up of one γ-chain and one Δ-chain. This group of T-cells is muchless common (2% of total T-cells) than the αβ T-cells.

“Central memory” T-cells (or “TCM”), as used herein, refers to anantigen experienced CTL that expresses CD62L or CCR-7 and CD45RO on thesurface thereof, and does not express or has decreased expression ofCD45RA as compared to naive cells. In particular embodiments, centralmemory cells are positive for expression of CD62L, CCR7, CD2S, CD127,CD45RO, and CD95, and have decreased expression of CD54RA as compared tonaive cells.

“Effector memory” T-cell (or “TEM”), as used herein, refers to anantigen experienced T-cell that does not express or has decreasedexpression of CD62L on the surface thereof as compared to central memorycells, and does not express or has decreased expression of CD45RA ascompared to a naive cell. In particular embodiments, effector memorycells are negative for expression of CD62L and CCR7, compared to naivecells or central memory cells, and have variable expression of CD28 andCD45RA.

“Naive” T-cells, as used herein, refers to a non-antigen experienced Tlymphocyte that expresses CD62L and CD45RA, and does not express CD45ROas compared to central or effector memory cells. In particularembodiments, naive CD8+T lymphocytes are characterized by the expressionof phenotypic markers of naive T-cells including CD62L, CCR7, CD28,CD127, and CD45RA.

“Effector” or “TE” T-cells, as used herein, refers to an antigenexperienced cytotoxic T lymphocyte cells that do not express or havedecreased expression of CD62L, CCR7, CD28, and are positive for granzymeB and perforin as compared to central memory or naive T-cells.

NK cells are cytotoxic lymphocytes that can rapidly respond to viralinfection or tumor formation. NK cells can recognize “stressed” cells inthe absence of MHC expression or antibodies, and can release cytolyticgranules containing proteins such as perforin, which may form pores incell membranes of nearby cells. NK cells can become activated in thepresence of cytokines including IL-12, IL-15, IL-18, IL-2, and CCL5. NKcells may become activated upon ligand binding to an NK cell activatingreceptor. Receptors that can contribute to NK cell activation includeCD137, CD2, and CD44.

Each of the lymphocyte types described herein can be embedded in thescaffolds disclosed herein. In particular embodiments, the primarylymphocyte cell type will be CTL. CTLs can be included at 50% or more ofthe embedded lymphocyte population, 55% or more of the embeddedlymphocyte population, 60% or more of the embedded lymphocytepopulation, 65% or more of the embedded lymphocyte population, 70% ormore of the embedded lymphocyte population, 75% or more of the embeddedlymphocyte population, 80% or more of the embedded lymphocytepopulation, 85% or more of the embedded lymphocyte population, 90% ormore of the embedded lymphocyte population, 95% or more of the embeddedlymphocyte population, or 100% the embedded lymphocyte population.

Various combinations of lymphocytes can also be used in the scaffoldsdisclosed herein. In particular embodiments, the scaffold includes amixture of CD8+ cells, NK cells, invariant NKT cells (iNKT cells), Th17CD4+ cells and/or B cells. In particular embodiments, the scaffoldsinclude a mixture of CD8+ cells and NK cells. In particular embodiments,the mixture of CD8+ cells and NK cells is a 50:50 mix. In particularembodiments, the scaffolds include a mixture of CD8+ cells and iNKTcells. In particular embodiments, the mixture of CD8+ cells and iNKTcells is a 50:50 mix. All other possible combinations of the disclosedcell types can also be used within the scaffolds disclosed herein.

In particular embodiments, the lymphocytes can be isolated and expandedfrom resected tumor. In particular embodiments, subjects can bevaccinated with a tumor antigen (e.g., against Her2) and vaccine-inducedT-cell populations can be expanded and embedded into the scaffold.

Lymphocytes within the scaffolds can be non-genetically modified orgenetically-modified or can be provided in a combination ofnon-genetically-modified and genetically-modified forms. Geneticmodifications can be made to enhance growth, survival, immune functionand/or tumor cell targeting. Examples of genetic modifications includethose allowing expression of: a chimeric antigen receptor (CAR), a αβT-cell receptor (or modification thereof), and/or pro-inflammatorycytokines; or blocking expression of an inhibitor signal (e.g.,killer-cell immunoglobulin-like receptor). CAR modification and/or αβT-cell receptor modifications allow the modified lymphocytes tospecifically target cell types.

In one aspect, genetically-modified lymphocytes can have improved tumorrecognition, trigger increased native T-cell proliferation and/orcytokine production. Different potential CAR nucleic acid constructsthat encode different ligand binding domains, different spacer regionlengths, different intracellular binding domains and/or differenttransmembrane domains, can be tested in vivo (in an animal model) and/orin vitro to identify CARs with improved function over non-geneticallymodified lymphocytes and/or other CARs and in particular embodiments,using the scaffolds disclosed herein as an in vivo screening tool.

Exemplary CARs express ligand binding domains targeting, for example,NKG2D ligands, mesothelin, Her2, WT-1 and/or EGRF. An exemplary T-cellreceptor modification targets melanoma-associated antigen (MAGE) A3 TCR.

The particular following cancers can be targeted by including within anextracellular component of a TCR or CAR a binding domain that binds theassociated cellular marker(s):

Targeted Cancer Cellular Marker(s) Prostate Cancer PSMA, WT1, ProstateStem Cell antigen (PSCA), SV40 T Breast Cancer HER2, ERBB2, ROR1 StemCell Cancer CD133 Ovarian Cancer L1-CAM, extracellular domain of MUC16(MUC-CD), folate binding protein (folate receptor), Lewis Y, ROR1,mesothelin, WT-1 Mesothelioma mesothelin Renal Cell carboxy-anhydrase-IX(CAIX); Carcinoma Melanoma GD2 Pancreatic Cancer mesothelin, CEA, CD24,ROR1, NKG2D ligands (e.g., Rae-1) Lung Cancer ROR1

Without limiting the foregoing, cellular markers also include A33; BAGE;Bcl-2; β-catenin; B7H4; BTLA; CA125; CA19-9; CD3, CD5; CD19; CD20; CD21;CD22; CD25; CD28; CD30; CD33; CD37; CD40; CD52; CD44v6; CD45; CD56;CD79b; CD80; CD81; CD86; CD123; CD134; CD137; CD151; CD171; CD276; CEA;CEACAM6; c-Met; CS-1; CTLA-4; cyclin B1; DAGE; EBNA; EGFR; EGFRvIII,ephrinB2; ErbB2; ErbB3; ErbB4; EphA2; estrogen receptor; FAP; ferritin;α-fetoprotein (AFP); FLT1; FLT4; folate-binding protein; Frizzled; GAGE;G250; GD-2; GHRHR; GHR; GITR; GM2; gp75; gp100 (Pmel 17); gp130; HLA;HER-2/neu; HPV E6; HPV E7; hTERT; HVEM; IGF1R; IL6R; KDR; Ki-67; LewisA; Lewis Y; LIFRβ; LRP; LRP5; LTβR; MAGE; MART; mesothelin; MUC; MUC1;MUM-1-B; myc; NYESO-1; O-acetyl GD-2; O-acetyl GD3; OSMRβ; p53; PD1;PD-L1; PD-L2; PRAME; progesterone receptor; PSA; PSMA; PTCH1; RANK; ras;Robo1; RORI; survivin; TCRα; TCRβ; tenascin; TGFBR1; TGFBR2; TLR7; TLR9;TNFR1; TNFR2; TNFRSF4; TWEAK-R; TSTA tyrosinase; VEGF; and WT1.

Particular cancer cell cellular markers include:

Cancer SEQ Antigen Sequence ID NO: PSMAMWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKS  6SNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVK RQIYVAAFTVQAAAETLSEVAPSCA MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLG  7EQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQL MesothelinMALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEA  8APLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTHFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSVQEALSGTPCLLGPGPVLTVLALLLASTLA CD19MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDG  9PTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLASWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR CD20MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSF 10FMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPP QDQESSPIENDSSP ROR1MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSS 11WNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL WT1MGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVRSASETSEKR 12PFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL

In particular embodiments, ROR1-specific and CD19-specific CARs can beconstructed using VL and VH chain segments of the 2A2, R12, and R11 mAhs(ROR1) and FMC63 mAb (CD19). Variable region sequences for R11 and R12are provided in Yang et al, Plos One 6(6):e21018, Jun. 15, 2011. EachscFV can be linked by a (Gly₄Ser)₃ (SEQ ID NO: 13) protein to a spacerdomain derived from IgG4-Fc (UniProt Database: P01861, SEQ ID NO: 14)including either ‘Hinge-CH2-CH3’ (229 AA, SEQ ID NO: 15), ‘Hinge-CH3’(119 AA, SEQ ID NO: 16) or ‘Hinge’ only (12 AA, SEQ ID NO: 17)sequences. All spacers can contain a S→P substitution within the ‘Hinge’domain located at position 108 of the native IgG4-Fc protein, and can belinked to the 27 AA transmembrane domain of human CD28 (SEQ ID NO: 18,for an exemplary full-length CD28 see UniProt: P10747) and to aneffector domain signaling module including either (i) the 41 AAcytoplasmic domain of human CD28 with an LL→GG substitution located atpositions 186-187 of the native CD28 protein (SEQ ID NO: 19) or (ii) the42 AA cytoplasmic domain of human 4-1BB (UniProt: Q07011, SEQ ID NO:20), each of which can be linked to the 112 AA cytoplasmic domain ofisoform 3 of human CD3 (UniProt: P20963, SEQ ID NO: 21). The constructencodes a T2A ribosomal skip element (SEQ ID NO: 22)) and a tEGFRsequence (SEQ ID NO: 23) downstream of the chimeric receptor. tEGFR canbe replaced or supplemented with a tag cassette binding a sequence, suchas STREP-TAG® II (SEQ ID NO: 24; IBA GMBH Ltd., Gottingen, Del.), Myctag (SEQ ID NO: 25), V5 tag (SEQ ID NO: 26), FLAG® (Sigma-Aldrich, St.Louis, Mo.) tag (SEQ ID NO: 27), His tag, or other peptides or moleculesas disclosed herein. Codon-optimized gene sequences encoding eachtransgene can be synthesized (Life Technologies) and cloned into theepHIV7 lentiviral vector using NheI and Not1 restriction sites. TheepHIV7 lentiviral vector can be derived from the pHIV7 vector byreplacing the cytomegalovirus promoter of pHIV7 with an EF-1 promoter.ROR1-chimeric receptor, CD19-chimeric receptor, tEGFR, or tagcassette-encoding lentiviruses can be produced in 293T cells using thepackaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G, and Calphostransfection reagent (Clontech).

HER2-specific chimeric receptors can be constructed using VL and VHchain segments of a HER2-specific mAb that recognizes a membraneproximal epitope on HER2, and the scFVs can be linked to IgG4hinge/CH2/CH3, IgG4 hinge/CH3, and IgG4 hinge only extracellular spacerdomains and to the CD28 transmembrane domain, 4-1BB and CD3 signalingdomains.

As indicated, each CD19 chimeric receptor can include a single chainvariable fragment corresponding to the sequence of the CD19-specific mAbFMC63 (scFv: VL-VH), a spacer derived from IgG4-Fc including either the‘Hinge-CH2-CH3’ domain (229 AA, long spacer) or the ‘Hinge’ domain only(12 AA, short spacer), and a signaling module of CD3 with membraneproximal CD28 or 4-1BB costimulatory domains, either alone or in tandem.The transgene cassette can include a truncated EGFR (tEGFR) downstreamfrom the chimeric receptor gene and be separated by a cleavable T2Aelement, to serve as a tag sequence for transduction, selection and invivo tracking for chimeric receptor-modified cells. tEGFR can bereplaced or supplemented with a tag cassette binding a ExoCBM, such asSTREP-TAG® II (SEQ ID NO: 24), Myc tag (SEQ ID NO: 25), V5 tag (SEQ IDNO: 26), FLAG® tag (SEQ ID NO: 27), His tag, or other peptides ormolecules as disclosed herein.

Other common features of engineered CARs such as spacers, intracellulardomains, costimulatory domains, and transmembrane domains are known tothose of skill in the art.

In particular embodiments it may be desired to introduce functionalgenes into the lymphocytes to allow for negative selection in vivo asdescribed by, for example, Lupton et al., Mol. and Cell Biol., 11:6(1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); seealso the publications PCT/US91/08442 and PCT/US94/05601 by Lupton et.al. describing the use of bifunctional selectable fusion genes derivedfrom fusing a dominant positive selectable marker with a negativeselectable marker. This can be carried out in accordance with knowntechniques (see, e.g., U.S. Pat. No. 6,040,177 at columns 14-17) orvariations thereof that will be apparent to those skilled in the artbased upon the present disclosure. For example, it is contemplated thatoverexpression of a stimulatory factor (for example, a lymphokine or acytokine) may be toxic to the treated subject. Therefore, it is withinthe scope of the disclosure to include gene segments that cause thecells of the disclosure to be susceptible to negative selection in vivo.By “negative selection” is meant that the infused cell can be eliminatedas a result of a change in the in vivo condition of the individual. Thenegative selectable phenotype may result from the insertion of a genethat confers sensitivity to an administered agent, for example, acompound. Negative selectable genes are known in the art, and include,inter alia the following: the Herpes simplex virus type I thymidinekinase (HSV-I TK) gene, which confers ganciclovir sensitivity; thecellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellularadenine phosphoribosyltransferase (APRT) gene, and bacterial cytosinedeaminase.

Desired genes can be introduced into the lymphocytes prior to embeddingin a scaffold disclosed herein. Genetic reprogramming of a cell caninclude, for example, insertion of a gene sequence, alteration of a genesequence, and/or deletion of a gene sequence. In particular embodiments,lymphocytes can be genetically-reprogrammed by introducing a vector forgenetic reprogramming into the lymphocytes. Particular embodiments candeliver nucleotides within a gene editing system. Gene editing systemsmodify or affect particular sequences of a cell's endogenous genome.Gene editing systems are useful for targeted genome editing, for examplegene disruption, gene editing by homologous recombination, and genetherapy to insert therapeutic genes at the appropriate chromosomaltarget sites with a human genome.

Particular embodiments utilize transcription activator-like effectornucleases (TALENs) as gene editing systems. TALENs refer to fusionproteins including a transcription activator-like effector (TALE) DNAbinding protein and a DNA cleavage domain. TALENs are used to edit genesand genomes by inducing double strand breaks (DSBs) in the DNA, whichinduce repair mechanisms in cells. Generally, two TALENs must bind andflank each side of the target DNA site for the DNA cleavage domain todimerize and induce a DSB. The DSB is repaired in the cell bynon-homologous end-joining (NHEJ) or by homologous recombination (HR)with an exogenous double-stranded donor DNA fragment.

As indicated, TALENs have been engineered to bind a target sequence of,for example, an endogenous genome, and cut DNA at the location of thetarget sequence. The TALEs of TALENs are DNA binding proteins secretedby Xanthomonas bacteria. The DNA binding domain of TALEs include ahighly conserved 33 or 34 amino acid repeat, with divergent residues atthe 12th and 13th positions of each repeat. These two positions,referred to as the Repeat Variable Diresidue (RVD), show a strongcorrelation with specific nucleotide recognition. Accordingly, targetingspecificity can be improved by changing the amino acids in the RVD andincorporating nonconventional RVD amino acids.

Examples of DNA cleavage domains that can be used in TALEN fusions arewild-type and variant FokI endonucleases. The FokI domain functions as adimer requiring two constructs with unique DNA binding domains for siteson the target sequence. The FokI cleavage domain cleaves within a fiveor six base pair spacer sequence separating the two inverted half-sites.

Particular embodiments utilize MegaTALs as gene editing systems.MegaTALs have a single chain rare-cleaving nuclease structure in which aTALE is fused with the DNA cleavage domain of a meganuclease.Meganucleases, also known as homing endonucleases, are single peptidechains that have both DNA recognition and nuclease function in the samedomain. In contrast to the TALEN, the megaTAL only requires the deliveryof a single peptide chain for functional activity.

Particular embodiments utilize zinc finger nucleases (ZFNs) as geneediting systems. ZFNs are a class of site-specific nucleases engineeredto bind and cleave DNA at specific positions. ZFNs are used to introduceDSBs at a specific site in a DNA sequence which enables the ZFNs totarget unique sequences within a genome in a variety of different cells.Moreover, subsequent to double-stranded breakage, homologousrecombination or non-homologous end joining takes place to repair theDSB, thus enabling genome editing.

ZFNs are synthesized by fusing a zinc finger DNA-binding domain to a DNAcleavage domain. The DNA-binding domain includes three to six zincfinger proteins which are transcription factors. The DNA cleavage domainincludes the catalytic domain of, for example, FokI endonuclease.

Guide RNA can be used, for example, with gene-editing systems such asCRISPR-Cas systems. CRISPR-Cas systems include CRISPR repeats and a setof CRISPR-associated genes (Cas).

In general, any system capable of resulting in functional expression ofdelivered nucleotides can be used within the current disclosure.

Introduction of genes can be carried out by any method known in the art,including transfection, electroporation, microinjection, lipofection,calcium phosphate mediated transfection, infection with a viral orbacteriophage vector (e.g., a lentiviral vector or plasmid) containingthe gene sequences, cell fusion, chromosome-mediated gene transfer,microcell-mediated gene transfer, sheroplast fusion, etc. Numeroustechniques are known in the art for the introduction of foreign genesinto cells (see e.g., Loeffler and Behr, Meth. Enzymol, 217, 599-618(1993); Cohen et al., Meth. Enzymol, 217, 618-644 (1993); Cline,Pharmac. Ther, 29, 69-92 (1985)) and may be used in accordance with thepresent disclosure, provided that the necessary developmental andphysiological functions of the lymphocytes are not disrupted. Inparticular embodiments, the technique provides for the stable transferof the gene to the cell, so that the gene is expressible by the cell andpreferably heritable and expressible by its cell progeny. In particularembodiments, the technique provides for transient expression of the genewithin a cell.

Methods commonly known in the art of recombinant DNA technology whichcan be used to genetically modify the lymphocytes are described inAusubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression,A Laboratory Manual, Stockton Press, NY.

In particular embodiments, lymphocytes will be embedded within thescaffolds at or near the time of scaffold implantation in a subject, forexample within 48 hours of implantation, within 36 hours ofimplantation, within 24 hours of implantation, within 12 hours ofimplantation, within 6 hours of implantation, within 3 hours ofimplantation, within 1 hour of implantation or within 30 minutes ofimplantation. Generally, lymphocyte loading into pre-molded scaffoldswill occur within 30 minutes of implantation whereas the loading willmore often occur closer (i.e., within 5 minutes; within 2 minutes,within 1 minute or within 30 seconds) to the actual implantation timewhen injectable forms of the scaffolds are used.

The lymphocytes can be fresh lymphocytes or can be previouslycryo-preserved lymphocytes. If previously-cryopreserved lymphocytes areused, they should be thawed quickly (e.g., in a water bath maintained at37°-41° C.) and chilled immediately upon thawing. It may be desirable tofurther treat the lymphocytes in order to prevent cellular clumping uponthawing. To prevent clumping, various procedures can be used, includingthe addition before and/or after freezing of DNase, low molecular weightdextran and citrate, hydroxyethyl starch, etc. Where necessary due topotential cytotoxicities, cryoprotective agents should be removed. Afterremoval of cryoprotective agents, when necessary, cell count and/orviability testing can be performed.

A variety of methods to embed the lymphocytes into structures disclosedherein can be used (“embedding” is also referred to as “seeding”). Forexample, passive (static) seeding can be used. In particularembodiments, lymphocytes are resuspended in cell culture medium (e.g.,RPMI). This cell suspension is then added dropwise on top of alyophilized scaffold. In particular embodiments, where static seeding isused, a lymphocyte suspension is seeded onto a structure and afterwardsincubated for a certain time in the absence of agitation before beingexposed to dynamic culture conditions, for example into a spinner flaskthat is slowly agitated. In particular embodiments, dynamic seeding canbe used. For dynamic seeding the structure and the lymphocyte suspensioncan be placed together in, e.g., a container and the container is thenincubated with gentle agitation for a certain time allowing thelymphocytes to embed themselves within the structure. In additionalembodiments, rotational systems (including centrifuges) and/or vacuumsystems can be used. In additional embodiments, sheet-based lymphocyteseeding, electrostatic lymphocyte seeding, magnetic lymphocyte seeding,filtration lymphocyte seeding, and/or oscillating perfusion lymphocyteseeding can be used. Various combinations of these methods can also beused. The use of various biological hydrogels is also appropriate. Fordiscussions of the various seeding options, see Li et al., Biotechnol.Prog, 17, 935-944 (2001).; Wendt et al., Biotechnology andBioengineering, 84, 205-214 (2003); Yang, et al., J. Biomed. Mater. Res,55, 379-386 (2001); and Sittinger et al., Int. J. Artif. Organs, 20, 57(1997).

In particular embodiments, a lymphocyte scaffold including a TFNmicromesh can include a high-density of cells (at least 7×10⁶ per cm² orat least 8×10⁶ cells per cm²). TFN micromesh can allow for high celldensities, for example, by packing three layers of cells into each layerof TFN micromesh (see, e.g., FIG. 10D). Stacking multiple layers of amicropatterned metallic thin film (e.g., TFN micromesh) can also beuseful for achieving high densities of cells.

Effective variants of proteins and protein sequences disclosed hereincan also be used. Variants include peptides having one or moreconservative amino acid substitutions. As used herein, a “conservativesubstitution” involves a substitution of one amino acid for anotherfound in one of the following conservative substitutions groups: Group1: Alanine (Ala), Glycine (Gly), Serine (Ser), Threonine (Thr); Group 2:Aspartic acid (Asp), Glutamic acid (Glu); Group 3: Asparagine (Asn),Glutamine (Gin); Group 4: Arginine (Arg), Lysine (Lys), Histidine (His);Group 5: Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine(Val); and Group 6: Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan(Trp).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function or chemical structure or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, Gly, Ala,Val, Leu, and Ile. Other groups containing amino acids that areconsidered conservative substitutions for one another include:sulfur-containing: Met and Cysteine (Cys); acidic: Asp, Glu, Asn, andGln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser,Thr, Pro, and Gly; polar, negatively charged residues and their amides:Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg,and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, andCys; and large aromatic residues: Phe, Tyr, and Trp. Additionalinformation is found in Creighton (1984) Proteins, W.H. Freeman andCompany.

Variants also include sequences with at least 70% sequence identity, 80%sequence identity, 85% sequence, 90% sequence identity, 95% sequenceidentity, 96% sequence identity, 97% sequence identity, 98% sequenceidentity, or 99% sequence identity to any of SEQ ID NOs: 1-27.

“% identity” refers to a relationship between two or more proteinsequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenproteins as determined by the match between strings of such sequences.“Identity” (often referred to as “similarity”) can be readily calculatedby known methods, including those described in: Computational MolecularBiology (Lesk, A. M., ed.) Oxford University Press, N Y (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, N Y (1994); Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., eds.) Humana Press, N J (1994);Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) AcademicPress (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,J., eds.) Oxford University Press, NY (1992). Preferred methods todetermine identity are designed to give the best match between thesequences tested. Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the Lasergene bioinformatics computing suite (DNASTAR®, Inc.,Madison, Wis.). Multiple alignment of the sequences can also beperformed using the Clustal method of alignment (Higgins and SharpCABIOS, 5, 151-153 (1989), with default parameters (GAP PENALTY=10, GAPLENGTH PENALTY=10). Relevant programs also include the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol.215:403-410 (1990)); DNASTAR®; and the FASTA program incorporating theSmith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc.Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.Publisher: Plenum, New York, N.Y.). Within the context of thisdisclosure it will be understood that where sequence analysis softwareis used for analysis, the results of the analysis are based on the“default values” of the program referenced. As used herein “defaultvalues” will mean any set of values or parameters which originally loadwith the software when first initialized.

The current disclosure also provides salts, solvates, hydrates,N-oxides, prodrugs, and/or active metabolites of molecules and/orpeptides described herein. Suitable acid addition salts can be preparedfrom an inorganic acid or an organic acid, in particularpharmaceutically acceptable organic acid. Examples of such inorganicacids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric and phosphoric acid. Appropriate organic acids can be selectedfrom aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids.

Suitable base addition salts can be prepared from a metallic salt or anorganic salt. Metallic salts can be prepared from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc. Organic salts can beprepared from N,N′-dibenzylethylene-diamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine,procaine, and any pharmaceutically acceptable organic bases.

Methods of Use. The scaffolds described herein can be placed in thevicinity of a solid tumor, an un-resecatable tumor and/or non-resectedtumor cells to have an anti-tumor effect in a subject. As used herein,the terms “subject” or “individual” typically refer to a mammal, such asa human, but can also be another mammal such as dogs, cats, rabbits,cows, horses, etc.

A “tumor” is a swelling or lesion formed by an abnormal growth of cells(called neoplastic cells or tumor cells). A “tumor cell” is an abnormalcell that divides by a rapid, uncontrolled cellular proliferation andcontinues to divide after the stimuli that initiated the new divisioncease. Tumors show partial or complete lack of structural organizationand functional coordination with the normal tissue, and usually form adistinct mass of tissue, which may be either benign, pre-malignant ormalignant.

As used herein, an anti-tumor effect refers to a biological effect,which can be manifested by a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or a decrease of various physiologicalsymptoms associated with the cancerous condition. An anti-tumor effectcan also be manifested by a decrease in recurrence or an increase in thetime before recurrence. Accordingly, the scaffolds disclosed herein canbe used to treat a variety of cancers, can prevent or significantlydelay metastasis, and/or can prevent or significantly delay relapse. Asshown in FIGS. 9A and 9B, lymphocyte scaffolds can continue to provideanti-tumor immunity after eradication/reduction of an initial tumor, andcan prevent and/or reduce metastases or development of secondary tumors.

Cancer (medical term: malignant neoplasm) refers to a class of diseasesin which a group of cells display uncontrolled growth (division beyondthe normal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis. “Metastasis” refers to the spread ofcancer cells from their original site of proliferation to another partof the body. The formation of metastasis is a very complex process anddepends on detachment of malignant cells from the primary tumor,invasion of the extracellular matrix, penetration of the endothelialbasement membranes to enter the body cavity and vessels, and then, afterbeing transported by the blood, infiltration of target organs. Finally,the growth of a new tumor, i.e. a secondary tumor or metastatic tumor,at the target site depends on angiogenesis. Tumor metastasis oftenoccurs even after the removal of the primary tumor because tumor cellsor components may remain and develop metastatic potential.

Cancers that can be treated with the anti-tumor effects of the scaffoldsand methods disclosed herein include, for example, adrenal cancer, braincancer, breast cancer, cervical cancer, colon cancer, colorectal cancer,ear, nose and throat (ENT) cancer, endometrial cancer, esophagealcancer, gastrointestinal cancer, gliomas, head and neck cancer,intestinal cancer, kidney cancer, liver cancer, lung cancer, lymph nodecancer, melanomas, neuroblastomas, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, seminomas, skin cancer, stomach cancer,teratomas, thyroid cancer, uterine cancer, and metastases thereof.

Without limiting the scope of the disclosure, the following cancer typesare noted:

Brain tumor (Glioblastoma): An estimated 10,000 new cases/year in theU.S. are seen. Currently no curative therapy is available. Gliobastomashows very infiltrative growth and cannot be resected completely. 90% oftumors relapse within a 2 cm margin from the originally resected tumor.Biomaterial wafers loaded with chemotherapy are United States Food andDrug Administration (FDA)-approved (GLIADEL®, MGI Pharma, Inc.,Woodcliff Lake, N.J.) for glioblastoma. However, due to insufficienttissue penetration, biomaterial implant delivered chemotherapy is mostlyineffective. In contrast, tumor-reactive lymphocytes deployed from thescaffolds disclosed herein can actively migrate to affected tissue,seeking out and destroying residual tumor cells.

Pancreatic adenocarcinoma: An estimated 43,920 new cases of pancreaticcancer were expected to occur in the U.S. in 2012. Only 20% will haveresectable disease at the time of diagnosis (80% of patients do notundergo surgery as their tumor is too advanced at the time ofdiagnosis). Even surgery is considered a palliative venture with a5-year survival rate of only 20%. Local recurrence is usually attributedto the difficulty of achieving microscopically negative surgicalmargins. Beyond the current scaffold's ability to eradicate residualdisease following surgical tumor resection, the scaffolds can alsoprovide pancreatic tumor patients with inoperable disease (80% ofpatients) with a highly effective treatment option. In particularembodiments, scaffolds are implanted directly onto un-resectableestablished pancreatic adenocarcinomas.

Ovarian cancer: An estimated 22,000 new cases in 2012 in the U.S. wereseen. Despite multimodality therapy with surgery and chemotherapy, mostovarian cancer patients have a poor prognosis (15,500 estimateddeaths/year in U.S.). Ovarian cancer primarily disseminates within theperitoneal cavity. Adoptive T-cell therapy in ovarian cancer patients iscurrently being investigated at several centers. However, to dateclinical results have been disappointing due to a poor survival ofinfused T-cells and a failure to combat immunosuppressive factorsreleased by tumor cells to render T-cells dysfunctional. Multiplescaffolds embedded with tumor-reactive lymphocytes could be implantedlaparoscopically into the peritoneal cavity of ovarian cancer patients,where they release tumor-reactive lymphocytes and STING agonists over anextended time period.

As will be understood by one of ordinary skill in the art, the scaffoldsare implanted in close proximity to a solid tumor, un-resectable tumorcells and/or in tumor resection beds following resection. The scaffoldscan be available in a number of different sizes and shapes and can beshape-conformable to fit the particular needs of individual subjects. Inparticular embodiments, the scaffolds are injected using ultrasoundguidance in close proximity to (or in physical contact with) a solidtumor, un-resected or non-resected tumor cells. Depending on the stage,size or severity of a tumor, scaffolds may be provided with differenttherapeutic strengths. Therapeutic strength can be manipulated byaltering the size of the scaffold, volume of the scaffold, the number oflymphocytes embedded within a scaffold, the number oflymphocyte-activating moieties within a scaffold, the presence or amountof STING agonists within the scaffold, etc. Each of these parameters canbe assessed and determined by a treating physician.

For the purposes of the present disclosure, the term “proximity” refersto a distance within 10 cm, within 9 cm, within 8 cm, within 7 cm,within 6 cm, within 5 cm, within 4 cm, within 3 cm, within 2 cm, within1 cm, within 0.9 cm, within 0.8 cm, within 0.7 cm, within 0.6 cm, within0.5 cm, within 0.4 cm, within 0.3 cm, within 0.2 cm, or within 0.1 cm ofa solid tumor, an un-resectable tumor, un-resectable tumor cells, and/ora tumor resection bed.

It is also understood by one of ordinary skill in the art that thescaffolds can be implanted only once, at the time of resection or at afirst treatment time in a subject with a solid tumor, an un-resectabletumor, un-resectable tumor cells, and/or a tumor resection bed.Additionally, the scaffolds can be implanted a plurality of times toprovide ongoing therapy over months or years. Such treatment regimenscan be determined by a treating physician.

In particular embodiments, a lymphocyte scaffold including a TFNmicromesh can be used as a long-acting scaffold (see, e.g., FIG. 12I).TFN micromesh can be used as a non-biodegradable scaffold matrixmaterial, and therefore a TFN-micromesh based lymphocyte scaffold maycontinue to deliver lymphocytes for several days, for more than oneweek, and/or for more than two weeks.

As used herein, the term “surgical treatment failure” refers to relapseof cancer in a subject who had previously undergone tumor resection.Surgical treatment failure may include metastatic relapse.

The Examples and Exemplary Embodiments below are included to demonstrateparticular embodiments of the disclosure. Those of ordinary skill in theart should recognize in light of the present disclosure that manychanges can be made to the specific embodiments disclosed herein andstill obtain a like or similar result without departing from the spiritand scope of the disclosure.

Exemplary Embodiments

1. A lymphocyte scaffold including (i) genetically-reprogrammedlymphocytes disposed within a scaffold matrix including a micropatternedmetallic thin film, and (ii) a lymphocyte-activating moiety.2. The lymphocyte scaffold of embodiment 1, further including a STINGagonist.3. The lymphocyte scaffold of embodiment 2, wherein the STING agonistincludes c-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP, (2′,2′)c-AIMP,(2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP), c-(dAMP-2′FdIMP),c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP), c-[2′FdAMP(S)-2′FdIMP(S)],c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/or DMXAA.4. The lymphocyte scaffold of embodiment 2, wherein the STING agonistincludes c-diGMP.5. The lymphocyte scaffold of any of embodiments 1-4, further includinga drug eluting polymer.6. The lymphocyte scaffold of embodiment 5, wherein the STING agonist isembedded within the drug eluting polymer.7. The lymphocyte scaffold of embodiment 5 or 6, wherein the drugeluting polymer includes PLGA.8. The lymphocyte scaffold of any of embodiments 1-7, wherein thelymphocytes include T-cells and/or natural killer cells.9. The lymphocyte scaffold of any of embodiments 1-8, wherein thelymphocytes include CD8+ T-cells.10. The lymphocyte scaffold of any of embodiments 1-9, including atleast 2×10⁶ lymphocytes.11. The lymphocyte scaffold of any of embodiments 1-9, including atleast 7×10⁶ lymphocytes.12. The lymphocyte scaffold of any of embodiments 1-11, wherein thelymphocyte-activating moiety includes at least one of IL-15, or anantibody specific for CD3, CD28, or CD137.13. The lymphocyte scaffold of any of embodiments 1-12, wherein thelymphocyte-activating moiety includes antibodies specific for CD3,CD128, and CD137.14. The lymphocyte scaffold of any of embodiments 1-13, wherein thelymphocyte-activating moiety includes IL-15 and CD137.15. The lymphocyte scaffold of any of embodiments 1-14, furtherincluding an immune stimulant.16. The lymphocyte scaffold of embodiment 15, wherein the immunestimulant is a cytokine, an antibody, a small molecule, an siRNA, aplasmid DNA, and/or a vaccine adjuvant.17. The lymphocyte scaffold of embodiment 15, wherein the immunestimulant is selected from (i) a Toll-like receptor ligand selected fromCpG, Cpg-28, Poly(I:C), α-galactoceramide, MPLA, VTX-2337, EMD1201081)imiquimod, MGN1703, G100, CBLB502, Hiltonol, and Imiquimod, and/or (ii)17-dimethylaminoethylamino-17-demethoxygeldanamycin).18. The lymphocyte scaffold of any of embodiments 15-17, wherein theimmune stimulant is embedded within a drug eluting polymer.19. The lymphocyte scaffold of any of embodiments 1-18, furtherincluding a lymphocyte-adhesion moiety.20. The lymphocyte scaffold of embodiment 19, wherein thelymphocyte-adhesion moiety and the lymphocyte-activating moiety arecovalently linked.21. The lymphocyte scaffold of embodiment 19 or 20, wherein thelymphocyte-adhesion moiety includes fibrin.22. The lymphocyte scaffold of any of embodiments 19-21, wherein thelymphocyte-adhesion moiety includes a peptide that binds α₁β₁ integrin,α2β₁ integrin, α4β₁ integrin, α5β₁ integrin, or lymphocyte functionassociated antigen (LFA-1).23. The lymphocyte scaffold of any of embodiments 19-22, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide.24. The lymphocyte scaffold of any of embodiments 19-23, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide ofSEQ ID NO: 1 or SEQ ID NO: 2.25. The lymphocyte scaffold of any of embodiments 19-24, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide.26. The lymphocyte scaffold of any of embodiments 19-25, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide of SEQ ID NO: 3.27. The lymphocyte scaffold of any of embodiments 19-26, wherein thelymphocyte-adhesion moiety includes a FNIII₇₋₁₀ peptide.28. The lymphocyte scaffold of any of embodiments 19-27, wherein thelymphocyte-adhesion moiety includes a FNIII₇₋₁₀ peptide of SEQ ID NO: 4.29. The lymphocyte scaffold of any of embodiments 2-28, wherein theSTING agonist and/or the lymphocyte-activating moiety is linked to thescaffold matrix.30. The lymphocyte scaffold of any of embodiments 2-29, wherein theSTING agonist and/or the lymphocyte-activating moiety is embedded withinthe scaffold matrix.31. The lymphocyte scaffold of any of embodiments 2-30, wherein theSTING agonist and/or the lymphocyte-activating moiety is within abioactive coating overlaying at least a portion of the surface of thescaffold matrix.32. The lymphocyte scaffold of embodiment 31, wherein the bioactivecoating includes a drug eluting polymer.33. The lymphocyte scaffold of embodiment 32, further including alymphocyte-adhesion moiety, wherein: the STING agonist is within thedrug eluting polymer, the drug eluting polymer forms a monolayer on thesurface of the scaffold matrix, and the lymphocyte-adhesion moietydirectly coats the drug eluting polymer.34. The lymphocyte scaffold of any of embodiments 2-33, wherein theSTING agonist and/or the lymphocyte-activating moiety is linked toparticles.35. The lymphocyte scaffold of embodiment 34, wherein the particles arelinked to the scaffold and/or embedded within the scaffold matrix.36. The lymphocyte scaffold of any of embodiments 2-35, wherein theSTING agonist and/or the lymphocyte-activating moiety is bound to aliposome of a protocell.37. The lymphocyte scaffold of embodiment 36, wherein the ratio of theprotocells to the lymphocytes within the scaffold matrix is 0.5:1; 1:1;5:1; or 10:1.38. The lymphocyte scaffold of any of embodiments 1-37, wherein thelymphocyte scaffold includes 7×10⁶ to 1×10¹⁰ protocells.39. The lymphocyte scaffold of any of embodiments 1-38, wherein themicropatterned metallic thin film includes a TFN micromesh.40. A lymphocyte scaffold consisting of a scaffold matrix, geneticallyreprogrammed lymphocytes, and three lymphocyte-activating moieties.41. The lymphocyte scaffold of embodiment 40, wherein the scaffoldmatrix includes an alginate scaffold, a collagen/alginate scaffold, achitosan scaffold, a self-assembling peptide scaffold, a mesoporoussilica scaffold, a micropatterned metallic thin film scaffold, or a PLGAscaffold.42. The lymphocyte scaffold of embodiment 40 or 41, wherein themicropatterned metallic thin film scaffold includes a TFN micromeshscaffold.43. The lymphocyte scaffold of any of embodiments 40-42, wherein thescaffold matrix includes an alginate scaffold.44. The lymphocyte scaffold of any of embodiments 40-43, wherein thescaffold matrix includes a polymeric calcium cross-linked alginatescaffold.45. The lymphocyte scaffold of any of embodiments 40-44, wherein thegenetically reprogrammed lymphocytes are genetically reprogrammedT-cells and/or natural killer cells.46. The lymphocyte scaffold of any of embodiments 40-45, wherein thegenetically reprogrammed lymphocytes are CD8+ T-cells.47. The lymphocyte scaffold of any of embodiments 40-46, including atleast 2×10⁶ genetically reprogrammed lymphocytes.48. The lymphocyte scaffold of any of embodiments 40-46, including atleast 7×10⁶ genetically reprogrammed lymphocytes.49. The lymphocyte scaffold of any of embodiments 40-48, wherein thelymphocyte-activating moieties include antibodies specific for CD3,CD28, and/or CD137.50. The lymphocyte scaffold of any of embodiments 40-49, wherein thelymphocyte-activating moieties are linked to the scaffold.51. The lymphocyte scaffold of any of embodiments 40-50, wherein thelymphocyte-activating moieties are embedded within the scaffold.52. A lymphocyte scaffold including: a scaffold matrix,genetically-reprogrammed lymphocytes, and a lymphocyte-activatingmoiety.53. The lymphocyte scaffold of embodiment 52, further including a STINGagonist.54. The lymphocyte scaffold of embodiment 53, wherein the STING agonistincludes c-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP, (2′,2′)c-AIMP,(2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP), c-(dAMP-2′FdIMP),c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP), c-[2′FdAMP(S)-2′FdIMP(S)],c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/or DMXAA.55. The lymphocyte scaffold of embodiment 52 or 53, wherein the STINGagonist includes c-diGMP.56. The lymphocyte scaffold of any of embodiments 52-55, wherein thegenetically-reprogrammed lymphocytes are genetically-reprogrammedT-cells and/or natural killer cells.57. The lymphocyte scaffold of any of embodiments 52-56, wherein thegenetically-reprogrammed lymphocytes are CD8+ T-cells.58. The lymphocyte scaffold of any of embodiments 52-57, including atleast 2×10⁶ or at least 7×10⁶ genetically-reprogrammed lymphocytes.59. The lymphocyte scaffold of any of embodiments 52-58, wherein thelymphocyte-activating moiety includes IL-15 and/or antibodies specificfor CD3, CD28, and/or CD137.60. The lymphocyte scaffold of any of embodiments 52-59, furtherincluding an immune stimulant.61. The lymphocyte scaffold of embodiment 60, wherein the immunestimulant includes a cytokine, an antibody, a small molecule, an siRNA,a plasmid DNA, and/or a vaccine adjuvant.62. The lymphocyte scaffold of embodiment 60 or 61, wherein the immunestimulant includes (i) a Toll-like receptor ligand selected from CpG,Cpg-28, Poly(I:C), α-galactoceramide, MPLA, VTX-2337, EMD1201081)imiquimod, MGN1703, G100, CBLB502, Hiltonol, and Imiquimod, and/or (ii)17-dimethylaminoethylamino-17-demethoxygeldanamycin).63. The lymphocyte scaffold of any of embodiments 52-62, furtherincluding a lymphocyte-adhesion moiety.64. The lymphocyte scaffold of embodiment 63, wherein thelymphocyte-adhesion moiety includes fibrin.65. The lymphocyte scaffold of embodiment 63 or 64, wherein thelymphocyte-adhesion moiety includes a peptide that binds α₁β₁ integrin,α2β₁ integrin, α4β₁ integrin, α5β₁ integrin, or lymphocyte functionassociated antigen (LFA-1).66. The lymphocyte scaffold of any of embodiments 63-65, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide.67. The lymphocyte scaffold of any of embodiments 63-66, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide ofSEQ ID NO: 1 or SEQ ID NO: 2.68. The lymphocyte scaffold of any of embodiments 63-67, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide.69. The lymphocyte scaffold of any of embodiments 63-68, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide of SEQ ID NO: 3.70. The lymphocyte scaffold of any of embodiments 63-69, wherein thelymphocyte-adhesion moiety includes a FNIII₁₇₋₁₀ peptide.71. The lymphocyte scaffold of any of embodiments 63-70, wherein thelymphocyte-adhesion moiety includes a FNIII₇₋₁₀ peptide of SEQ ID NO: 4.72. The lymphocyte scaffold of any of embodiments 53-71, wherein theSTING agonist and/or the lymphocyte-activating moiety is linked to thescaffold matrix.73. The lymphocyte scaffold of any of embodiments 53-72, wherein theSTING agonist and/or the lymphocyte-activating moiety is embedded withinthe scaffold matrix.74. The lymphocyte scaffold of any of embodiments 53-73, wherein theSTING agonist and/or the lymphocyte-activating moiety is within abioactive coating overlaying at least a portion of the surface of thescaffold matrix.75. The lymphocyte scaffold of any of embodiments 53-74, wherein theSTING agonist and/or the lymphocyte-activating moiety is linked toparticles.76. The lymphocyte scaffold of embodiment 75, wherein the particles arelinked to the scaffold matrix and/or embedded within the scaffoldmatrix.77. The lymphocyte scaffold of any of embodiments 53-76, wherein theSTING agonist and/or the lymphocyte-activating moiety is bound to aliposome of a protocell.78. The lymphocyte scaffold of embodiment 77, wherein the ratio of theprotocells to the lymphocytes within the scaffold matrix is 0.5:1; 1:1;5:1; or 10:1.79. The lymphocyte scaffold of any of embodiments 53-78, wherein thelymphocyte scaffold includes 7×10⁶ to 1×10¹⁰ protocells.80. The lymphocyte scaffold of any of embodiments 52-79, wherein thescaffold matrix includes an alginate scaffold, a collagen/alginatescaffold, a chitosan scaffold, a self-assembling peptide scaffold, amesoporous silica scaffold, a micropatterned metallic thin filmscaffold, or a PLGA scaffold.81. The lymphocyte scaffold of embodiment 80, wherein the micropatternedmetallic thin film scaffold includes a TFN micromesh scaffold.82. The lymphocyte scaffold of embodiment 80 or 81, wherein the scaffoldmatrix includes an alginate scaffold.83. The lymphocyte scaffold of any of embodiments 80-82, wherein thescaffold matrix includes a polymeric calcium cross-linked alginatescaffold.84. A lymphocyte scaffold including: (i) a scaffold matrix material,(ii) natural killer cells with anti-cancer activity, and (iii)lymphocyte-activating moieties including IL-15 and an antibody specificto CD137.85. The lymphocyte scaffold of embodiment 84, wherein the scaffoldmatrix material includes a micropatterned metallic thin film.86. The lymphocyte scaffold of embodiment 84 or 85, wherein themicropatterned metallic thin film includes a TFN micromesh.87. The lymphocyte scaffold of any of embodiments 84-86, furtherincluding a STING agonist.88. The lymphocyte scaffold of embodiment 113, wherein the STING agonistincludes c-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP, (2′,2′)c-AIMP,(2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP), c-(dAMP-2′FdIMP),c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP), c-[2′FdAMP(S)-2′FdIMP(S)],c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/or DMXAA.89. The lymphocyte scaffold of any of embodiments 84-88, furtherincluding a drug-eluting polymer.90. The lymphocyte scaffold of embodiment 89, wherein the drug elutingpolymer includes PLGA.91. The lymphocyte scaffold of embodiment 87 or 88 wherein the STINGagonist is embedded within a drug eluting polymer.92. The lymphocyte scaffold of any of embodiments 84-91, furtherincluding a lymphocyte-adhesion moiety.93. The lymphocyte scaffold of embodiment 92, wherein thelymphocyte-adhesion moiety includes fibrin.94. The lymphocyte scaffold of embodiment 92 or 93, wherein thelymphocyte-adhesion moiety includes a peptide that binds α₁β₁ integrin,α2β₁ integrin, α4β₁ integrin, α5β₁ integrin, or lymphocyte functionassociated antigen (LFA-1).95. The lymphocyte scaffold of any of embodiments 92-94, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide.96. The lymphocyte scaffold of any of embodiments 92-95, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide ofSEQ ID NO: 1 or SEQ ID NO: 2.97. The lymphocyte scaffold of any of embodiments 84-96, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide.98. The lymphocyte scaffold of any of embodiments 84-97, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide of SEQ ID NO: 3.99. The lymphocyte scaffold of any embodiments 84-98, wherein thelymphocyte-adhesion moiety includes a FNIII₁₇₋₁₀ peptide.100. The lymphocyte scaffold of any of embodiments 84-99, wherein thelymphocyte-adhesion moiety includes a FNIII₇₋₁₀ peptide of SEQ ID NO: 4.101. A method of treating a heterogenous solid tumor including escapevariant tumor cells in a subject including implanting a lymphocytescaffold of any of embodiments 1-100 into the subject within a proximityof the heterogenous solid tumor cell sufficient to lead to thedestruction of the heterogenous solid tumor in the subject, therebytreating the heterogenous solid tumor including escape variant tumorcells.102. A method of vaccinating a subject against development of cancerrecurrence including implanting a lymphocyte scaffold of any ofembodiments 1-100 into the subject within a proximity of a heterogenoussolid tumor or within a solid tumor resection bed in the subject,thereby vaccinating the subject against development of cancerrecurrence.103. A method of treating tumor cells in a subject in need thereofincluding implanting a lymphocyte scaffold of any of embodiments 1-100into the subject within a tumor resection bed thereby treating the tumorcells in the subject.104. The method of embodiment 103, wherein a treated tumor cell is anadrenal cancer cell, a brain cancer cell, a breast cancer cell, acervical cancer cell, a colon cancer cell, a colorectal cancer cell, anear, nose and throat (ENT) cancer cell, an endometrial cancer cell, anesophageal cancer cell, a gastrointestinal cancer cell, a glioma cell, ahead and neck cancer cell, an intestinal cancer cell, a kidney cancercell, a liver cancer cell, a lung cancer cell, a lymph node cancer cell,a melanoma cell, a neuroblastoma cell, an ovarian cancer cell, apancreatic cancer cell, a prostate cancer cell, a rectal cancer cell, aseminoma cell, a skin cancer cell, a stomach cancer cell, a teratomacell, a thyroid cancer cell, or a uterine cancer cell.105. The method of embodiment 103, wherein a treated tumor cell is aglioblastoma cell, a pancreatic adenocarcinoma cell, or an ovariancancer cell.106. A method of reducing surgical treatment failure caused bymetastatic relapse after resection of a primary tumor, includingadministering a lymphocyte scaffold of any of embodiments 1-100 to atumor resection bed of a subject thereby reducing surgical treatmentfailure caused by metastatic relapse after primary tumor resection.107. The method of embodiment 106, wherein the primary tumor includes aseminoma cell, a melanoma cell, a teratoma cell, a neuroblastoma cell, aglioma cell, a rectal cancer cell, an endometrial cancer cell, a kidneycancer cell, an adrenal cancer cell, a thyroid cancer cell, a skincancer cell, a brain cancer cell, a cervical cancer cell, an intestinalcancer cell, a liver cancer cell, a colon cancer cell, a stomach cancercell, a head and neck cancer cell, a gastrointestinal cancer cell, alymph node cancer cell, an esophageal cancer cell, a colorectal cancercell, a pancreatic cancer cell, an ear, nose and throat (ENT) cancercell, a breast cancer cell, a prostate cancer cell, a uterine cancercell, an ovarian cancer cell, or a lung cancer cell.108. A method of treating a subject for cancer including implanting intothe subject a medical device coated with a lymphocyte scaffold includinga TFN micromesh, genetically-modified lymphocytes, and a lymphocyteactivating moiety.109. The method of embodiment 108, wherein the implanting includes aminimally invasive procedure.110. The method of embodiment 108, wherein the medical device includes astent.111. A kit to form a lymphocyte scaffold to treat a solid tumor in asubject including (i) a scaffold matrix; and (ii) lymphocyte-activatingmoieties including antibodies specific for CD3, CD28, and CD137.112. The kit of embodiment 111, further including porous particles.113. The kit of embodiment 111 or 112, further including liposomes.114. The kit of any of embodiments 111-113, further includingprotocells.115. The kit of any of embodiments 111-114, further including a STINGagonist.116. The kit of embodiment 115, wherein the STING agonist includesc-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP, (2′,2′)c-AIMP,(2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP), c-(dAMP-2′FdIMP),c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP), c-[2′FdAMP(S)-2′FdIMP(S)],c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/or DMXAA.117. The kit of embodiment 115, wherein the STING agonist includesc-diGMP.118. The kit of any of embodiments 111-117, further including an immunestimulant.119. The kit of embodiment 118, wherein the immune stimulant includes acytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and/ora vaccine adjuvant.120. The kit of embodiment 118 or 119, wherein the immune stimulantincludes (i) a Toll-like receptor ligand selected from CpG, Cpg-28,Poly(I:C), α-galactoceramide, MPLA, VTX-2337, EMD1201081) imiquimod,MGN1703, G100, CBLB502, Hiltonol, and Imiquimod, and/or (ii)17-dimethylaminoethylamino-17-demethoxygeldanamycin).121. The kit of any of embodiments 111-120, further including alymphocyte-adhesion moiety.122. The kit of embodiment 121, wherein the lymphocyte-adhesion moietyincludes fibrin.123. The kit of embodiment 121 or 122, wherein the lymphocyte-adhesionmoiety includes a peptide that binds α₁β₁ integrin, α2β₁ integrin, α4β₁integrin, α5β₁ integrin, or lymphocyte function associated antigen(LFA-1).124. The kit of any of embodiments 121-123, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide.125. The kit of any of embodiments 121-124, wherein thelymphocyte-adhesion moiety includes a GFOGER (SEQ ID NO: 1) peptide ofSEQ ID NO: 1 or SEQ ID NO: 2.126. The kit of any of embodiments 121-125, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide.127. The kit of any of embodiments 121-126, wherein thelymphocyte-adhesion moiety includes an ICAM-1 peptide of SEQ ID NO: 3.128. The kit of any of embodiments 121-127, wherein thelymphocyte-adhesion moiety includes a FNIII₁₇₋₁₀ peptide.129. The kit of any of embodiments 121-128, wherein thelymphocyte-adhesion moiety includes a FNIII₁₇₋₁₀ peptide of SEQ ID NO:4.130. The kit of any of embodiments 111-129, wherein the kit includes7×10⁶ to 1×10¹⁰ particles, liposomes or protocells.131. The kit of any of embodiments 111-130, further includinggenetically-reprogrammed lymphocytes.132. The kit of embodiment 131, wherein the genetically-reprogrammedlymphocytes include T-cells and/or natural killer cells.133. The kit of embodiment 131 or 132, wherein thegenetically-reprogrammed lymphocytes include CD8+ T-cells.134. The kit of any of embodiments 131-133, wherein the lymphocytesinclude at least 7×10⁶ lymphocytes.135. The kit of any of embodiments 131-134, wherein the scaffold matrixincludes alginate, collagen, chitosan, a self-assembling peptide,mesoporous silica, TFN micromesh, or PLGA.136. The kit of embodiment 135, wherein the scaffold matrix includesalginate.137. The kit of any of embodiments 111-136, further including calcium.138. The kit of any of embodiments 111-137, further including a drugeluting polymer.139. The kit of embodiment 138, wherein the drug eluting polymer iscoated on the scaffold matrix.140. The kit of embodiment 138, wherein a STING agonist or an immunestimulant is embedded within the drug eluting polymer.141. The kit of any of embodiments 111-140, wherein the drug elutingpolymer includes PLGA.142. An implantable medical device including: (i) a micropatternedmetallic thin film scaffold, (ii) genetically reprogrammed lymphocytes,and (iii) a lymphocyte-activating moiety.143. The implantable medical device of embodiment 142 further includinga STING agonist and/or an immune stimulant.144. The implantable medical device of embodiment 143 further includinga drug eluting polymer, wherein the STING agonist and/or the immunestimulant is embedded within the drug eluting polymer.145. The implantable medical device of any of embodiments 142-144,wherein the micropatterned metallic thin film scaffold has athree-dimensional shape.146. The implantable medical device of embodiment 145, wherein thethree-dimensional shape is a cylinder.147. The implantable medical device of any of embodiments 142-146,including a stent.148. The implantable medical device of any of embodiments 142-147,including a minimally invasive medical device.149. The implantable medical device of any of embodiments 142-148,wherein the micropatterned metallic thin film scaffold is stacked inlayers.150. The implantable medical device of any of embodiments 142-149,wherein the micropatterned metallic thin film includes a TFN micromesh.151. The implantable medical device of any of embodiments 142-150,wherein the genetically-reprogrammed lymphocytes are at a concentrationof at least 7×10⁶ cells/cm³.

Examples

Methods. Human pancreatic ductal adenocarcinoma. For the confocalimaging of human pancreatic adenocarcinoma (PDA, FIG. 1) fresh tumor wasprocured from patients undergoing pancreatic resection for PDA, whoprovided written informed consent under a research protocol approved bythe Cancer Consortium Institutional Review Board (CC-IRB) at the FredHutchinson Cancer Research Center.

Cell lines. The murine pancreatic ductal adenocarcinoma cell line KPC, agift from Dr. Sunil Hingorani (Fred Hutchinson Cancer Research Center,Seattle, Wash.), was derived from spontaneously developing pancreatictumors of transgenic KPC (LSL-KrasG12D; p53lox/+) mice at 17 weeks ofage. This cell line was cultured in IMDM medium with 10%heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1.5 g/Lsodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodiumpyruvate, and 0.05 mM 2-mercaptoethanol. The Phoenix™ Eco retroviralpackaging cell line (Orbigen) was cultured in DMEM containing 10% FBS, 2mM glutamate, 100 U/mL penicillin, and 100 μg/mL streptomycin. For invivo bioluminescent imaging, the KPC cell line was retrovirallytransduced with firefly luciferase (F-luc).

Mice and in vivo tumor model. Animals were housed in the animal facilityof Fred Hutchinson Cancer Research Center, and used in the context of ananimal protocol approved by their Institutional Animal Care and UseCommittee. For the orthotopic mouse model of pancreatic ductaladenocarcinoma, 1×10⁵ KPC tumor cells were surgically implanted into thehead of the pancreas in female Albino B6 (C57BL/6J-Tyr<c-2J>) mice(Jackson Laboratories) and were allowed to establish for one week beforetreatment. To differentiate between adoptively transferred andendogenous T cells in the flow cytometry studies shown in FIG. 7A, 7B,NKG2D CAR T cells were generated from splenocytes isolated fromwild-type (CD45.2+) C57BL/6 mice. Following gene transfer, T cells wereused to treat B6.SJL-Ptprca Pepcb/BoyJ recipient mice (JacksonLaboratories), which express the pan leukocyte marker CD45.1.

Retroviral vectors and viral production. SFG-CBR-luc (expressing clickbeetle red luciferase) and SFG-F-luc (expressing firefly luciferase)were kindly provided by Dr. Michel Sadelain (Memorial Sloan-KetteringCancer Center, New York). The retroviral vector pFb-chNKG2D-IRES-Neo wasprovided by Dr. Charles Sentman (The Geisel School of Medicine atDartmouth, Lebanon), and has been described previously. Zhang, et al.,Blood 10⁶, 1544-1551 (2005). NKG2D CARs include the full-length mouseNKG2D (UniProt ID No. 054709) fused with the cytoplasmic portion of CD3.To generate retroviral particles, Phoenix Eco cells (1.5×10⁶/10 cmculture plate) were transfected overnight with 10 μg of vector-DNA usingstandard calcium phosphate methods; the following day, they wereincubated in 10 mL fresh DMEM for an additional day before theretroviral supernatant was filtered (0.45-μm, Nalgene) and concentrated10-fold using Ultracel 100K membranes (Millipore).

Preparation of tumor-targeting lymphocytes. To generate pancreaticcancer-specific (NKG2D CAR-transduced) T cells, spleens of C57BL/6J micewere harvested, macerated over a filter, and resuspended in ACK lysingBuffer (Biosource). Effector CD8+ T cells were prepared by incubatingsplenocytes (3×10⁶/mL) in complete RPMI 1640 with 1 ng/mL interleukin-7(PeproTech) and 2 μg/mL Concavalin A (Calbiochem) at 37° C. Two dayslater, dead cells were removed by Ficoll gradient separation (GEHealthcare) and CD8+ cells were isolated using a mouse CD8 NegativeIsolation Kit (Stemcell Technologies). Introduction of the NKG2D CARinto T cells was performed by retroviral transduction. ConcentratedNKG2D CAR expressing retrovirus (1 mL) was preloaded onto six-wellnon-tissue culture treated dishes coated with RetroNectin (TakiraBio)and incubated at 37° C. for 1 hr. An equal volume of isolated T-cells(3×10⁶ cells/mL supplemented with 10 ng mIL-2/mL) was added andcentrifuged at 2000×g for 30 min). 6 hr after spinoculation, 1 mL offresh, prewarmed RPMI, containing 10 ng mIL-2 (PeproTech) was added. Twodays after infection, transduced primary T cells (0.5-1×10/mL) wereselected in RPMI-10 media containing G418 (0.5 mg/mL) plus 25 U/mLrecombinant human (rHu) IL-2 for an additional 3 days. Viable cells wereisolated using Histopaque-1083 (Sigma, St Louis, Mo.) and expanded for 2days without G418 before adoptive transfer. For bioluminescence T-cellimaging experiments, the targeted T cells were genetically tagged withclick beetle red luciferase (CBR-luc). Dobrenkov, et al., J Nucl Med 49,1162-1170 (2008). Six hours after this spinoculation, 1 mL of RPMIcontaining 50 IU IL-2 was added, and the transduced T cells were usedfor experiments 1 day later.

Preparation of stimulatory lipid-coated silica microspheres. Preparationof maleimide-functionalized lipid film. Lipid stock solutions wereformulated in chloroform. 140 mL DOPC (10 mg/mL), 30 mL DSPE-PEG(2000)maleimide (5 mg/mL), 150 mL cholesterol (5 mg/mL), and 50 mL 18:1 PEG2000-PE (5 mg/mL, all purchased from Avanti Polar Lipids) were combinedto attain a DOPC:DSPE-PEG(2000) maleimide:cholesterol:PEG 2000-PE massratio of 55:5:30:10 and 2.5 mg total lipid. Chloroform was evaporatedunder a stream of nitrogen and residual solvent was removed under vacuumovernight.

Amine modification of silica microparticles. 500 mg of spherical silicagel (15 μm particle diameter, 100 Å pore diameter, Sorbent Technologies)was suspended in 4 mL of a 25% solution of3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AEPTMS) inethanol then mixed gently at room temperature for 5 h. Unreacted AEPTMSwas removed by centrifugation (2 min, 1000×g) and decantation of thesupernatant. The amine-modified silica was washed with ethanol (4×2 mL)then air-dried for 2 days.

Loading of STING agonist into mesoporous silica microparticles. A 100mg/mL suspension of amine-modified silica was prepared in phosphatebuffered saline at pH 7.2 (PBS), and 360 μL of it was combined with 500μL of c-di-GMP (InvivoGen, 2 mg/mL in PBS), gently vortexed for 1 h,then diluted with 400 μL PBS.

Lipid adsorption on silica. 400 μL of the SiO₂/c-di-GMP suspension wasadded to a 2.5 mg batch of lipid film and vortexed for 15 s at 10 minintervals for a total of 1 h. The particles were pelleted at 3500×g for2 min, washed with PBS (2×1 mL), then resuspended in 250 μL PBS.

Antibody conjugation to lipid-coated particles. The hinge regiondisulfide bonds of anti-mouse CD3, CD28, and CD137 antibodies (BioXcell)were selectively reduced with dithiothreitol (DTT) as previouslydescribed. Kwong, et al., Cancer Res 73, 1547-1558 (2013). After DTT wasremoved with a desalting column, these mildly-reduced antibodies(anti-CD3: 200 μg; anti-CD28 and CD137: 400 μg) were added to 250 μLmaleimide-functionalized particles, vortexed briefly before rotation for2 h, and centrifuged at 3500×g for 2 min. The pellet was washed with PBS(2×1 mL), then suspended in 125 μL PBS.

Scaffold fabrication. Alginate scaffolds were produced from highmolecular weight (250 kDa) ultrapure sodium alginate powder (NovamatrixPronova UP MVG alginate) enriched in G blocks 60%) after it was oxidizedwith sodium periodate to create hydrolytically labile bonds, aspreviously described. Boontheekul, et al., Biomaterials 26, 2455-2465(2005).

Briefly, 200 μL of 0.25% sodium periodate was added dropwise to 10 mLaqueous 1% alginate, and stirred in the dark at 25° C. for 5 h beforethe reaction was quenched by stirring with equimolar ethylene glycol for30 min. The sample was dialyzed against deionized water for three daysusing membranes with a 3,500 molecular weight cut-off, then lyophilized.The oxidized alginate solution was reconstituted in a MES solution (0.1M MES, 0.3 M NaCl, pH 6.5) and covalently conjugated to thecollagen-mimetic peptide “GFOGER50” (obtained from the MIT Biopolymersfacility) using carbodiimide chemistry51: sulfo-NHS, EDC (both ThermoScientific), and the GFOGER (SEQ ID NO: 1) peptide were addedsequentially, and 24 h later the solution was again dialyzed (MWCO 20kDa dialysis membrane, Thermo Scientific) and lyophilized.

To make scaffolds, the alginate stock was reconstituted to 7 mL of a 2%w/v solution in PBS, and warmed to 55° C. before mixing with 7×10⁶stimulatory microspheres in aqueous suspension. Mild cross-linking wasinitiated by adding 1.4 mL of 0.1% (w/v) calcium chloride solution whilevortexing, then 700 μL was immediately transferred per 15 mm roundTeflon-coated mold to form 2 mm-thick scaffolds. These were frozen at−78° C. and lyophilized to yield porous matrices, which were stored at4° C. in a desiccator.

T cell seeding onto scaffolds. Following ex vivo expansion, mouse CD8+effector T cells specific for 4T1 breast tumor antigens or geneticallyengineered to express NKG2D-CAR were washed twice in PBS and resuspendedin non-supplemented RPMI medium at a concentration of 14×10⁶ cells/mL.After adding 5% AlgiMatrix Firming Buffer (Invitrogen), 500 μL of thiscell suspension was immediately inoculated on top of each lyophilizedscaffold in a 24-well tissue culture plate. Cells were allowed to infuseinto these matrices on ice for 30 min before implantation into the tumorresection cavity or the peritoneal cavity.

Cytotoxicity assays. In vitro cytotoxic activity of T cells was measuredusing standard ⁵¹Cr release assays as described elsewhere. Erskine, JVis Exp, e3683 (2012). Briefly, 4T1 breast tumor, ID8-VEGF ovarian tumoror B16F10 melanoma control tumor cells were labeled with ⁵¹Cr for 1 h at37° C., washed with RPMI containing 10% FCS, and resuspended in the samemedium at a concentration of 1×10⁵ tumor cells/mL. T cells were added tothe suspensions at varying effector-to-target cell ratios in 96-wellplates (final volume, 200 μL) and incubated for 4 h at 37° C., then 30μL of supernatant from each well was transferred into Lumaplate-96microplates (Packard Bioscience) for analysis with a Top Count NXTmicroplate scintillation counter (Packard Bioscience). Effector cellnumbers were calculated based on the total number of IFN-γ+CD8+ T cellsmeasured by flow cytometry using commercially available kits (R&DSystems).

In vivo bioluminescence and imaging. D-Luciferin (Xenogen) in PBS (15mg/mL) was used as a substrate for F-luc (imaging of 4T1 breast tumorand ID8-VEGF ovarian tumor) and CBR-luc47 (T-cell imaging).Bioluminescence images were collected with a Xenogen IVIS SpectrumImaging System (Xenogen, Alameda, Calif.). Living Image software version4.3.1 (Xenogen) was used to acquire (and later quantitate) the data 10min after intraperitoneal injection of D-luciferin into animalsanesthetized with 150 mg/kg of 2% isoflurane (Forane, BaxterHealthcare). Acquisition times ranged from 10 sec to 5 min.

Flow cytometry. Anti-recombinant Annexin V (used to quantify apoptoticcells) and other antibodies used with the FACSCanto Flow Cytometer (BDBiosciences) were purchased from eBioscience.

Confocal microscopy. To visualize scaffolds by confocal microscopy,Hilyte Fluor 647 (Anaspec) was conjugated to alginate using standardEDC/NHS chemistry, then mixed 1 part of the conjugate with 9 parts ofGFOGER (SEQ ID NO: 1) peptide-modified alginate to fabricate scaffoldsas described above. T cells were labeled with CellTracker Orange CMTMR(Invitrogen) immediately before seeding into these scaffolds. Three dayspost-implantation, the tumor resection bed was snap-frozen in OCT(Tissue-Tek) to produce cryosections, which were fixed with 2%paraformaldehyde, mounted in ProLong Gold Antifade reagent (Invitrogen),and imaged with a Zeiss LSM 780 NLO laser scanning confocal microscope.

Statistics. Statistical significance of measured differences in T cellmigration parameters was calculated using one-way ANOVA, followed byDunnett's comparison test. Pairwise differences in the bioluminescenttumor and T cell signals were analyzed at selected time points using theWilcoxon rank-sum test, and survival data was characterized using theLog-rank test. All statistical analyses were performed using GraphPadPrism software version 6.0.

Study approval. Experiments and handling of mice were conducted underfederal, state, and local guidelines under an IACUC protocol and withapproval from the Fred Hutchinson Cancer Research Center IACUC.

Results. Intravenous injections of tumor-reactive T cells fail to clearpancreatic ductal adenocarcinoma. In order to test the new immunotherapyapproach in a clinically relevant setting, described experiments wereperformed using a cell line derived from the spontaneous pancreatictumors that LSL-KrasG12D; p53lox/+(KPC) mice produce (Hingorani, et al.Cancer Cell 7, 469-483 (2005)); this allowed creation of animmunocompetent, orthotopic murine model of pancreatic cancer that hasrapid and predictable growth kinetics. The KPC cells were geneticallytagged with luciferase so that tumor burdens could be noninvasivelyquantified using bioluminescence imaging. When orthotopicallytransplanted into the pancreas of non-KPC littermates, these tumor cellsreproducibly develop into lesions that mimic human pancreatic cancer interms of genetic mutations, histologic appearance, and heterogeneity ofantigen/target expression (FIGS. 2A-2C).

It was first verified that conventional intravenous injections ofpancreatic cancer-specific lymphocytes fail to eradicate tumors in thisKPC model. To create these lymphocytes, mouse T cells were transducedwith a retrovirus encoding a chimeric natural killer receptor (includingNKG2D linked to the cytoplasmic signaling domain of CD330; FIG. 2D) thatis specific for Rae-1, a KPC antigen that is recognized by T cellsexpressing these receptors (FIGS. 2E, 2F). In order to track andquantify the in vivo migration and accumulation of the transferred Tcells in relation to KPC tumors, vectors for click beetle luciferasewere included in the plasmid. The results establish that, althoughintravenously infused T cells accumulate at high levels in the spleenand the liver, they inefficiently traffic to KPC tumor sites (FIG. 2G),yielding a modest 4-day survival advantage compared to untreated controlanimals (FIG. 2H). Furthermore, Rae-1 target antigen expression levelswere only slightly lower following infusion of CAR-T cells when comparedto control lymphocytes (FIG. 2I).

Delivery via bioactive carriers substantially improves T cell expansionand function at the tumor site, but antigen-negative tumor subtypesescape elimination by these cells. The results described above promptedexploration of the potential of using biomaterials for the localizeddelivery of tumor-reactive T cells to pancreatic tumor sites, and tocreate ways to sustain them. A method to embed cancer-fighting immunecells in a resorbable polymeric device that can be surgically implantedwhere a tumor has been excised, or upon one that is non-resectable (FIG.3A) was recently developed and is described in US2016/0008399 andStephan, et al., Nat Biotechnol 33, 97-101 (2015). Already at theirtarget site, the delivered lymphocytes begin eliminating cancer cellsimmediately. The porous scaffolds are created from polymerized alginate,a castable, naturally-occurring polysaccharide that has been approved bythe FDA for human use because of its exceptional biocompatibility andbiodegradability. Baldwin & Kiick, Biopolymers 94, 128-140 (2010). Toenable them to function as efficient delivery vehicles for active Tcells, these devices were augmented with migration-promotingmacromolecules (e.g., collagen-mimetic peptide) and stimulatory cues(e.g., embedded microparticles displaying anti-CD3, anti-CD28, and antiCD137 antibodies; FIG. 3A). Ten days after introducing theluciferase-expressing KPC tumor cells, scaffolds containing 7×10⁶NKG2D-CAR+ T cells were implanted directly on top of the resultingpancreatic tumors (FIG. 3B). A second group received the same dose ofcells injected directly into the tumor, and control mice received notreatment. Bioluminescence imaging was used to quantify tumor growthand, in parallel experiments, to track tissue distribution, expansion,and persistence of the lymphocytes. It was found that the CAR-T cellsinjected directly into pancreatic tumors persisted poorly in theimmunosuppressive microenvironment and only produced a temporary delayin disease progression (21 days compared to 14.5 days median overallsurvival in the untreated control group, FIGS. 4A-4D). By contrast, Tcells delivered from implanted scaffolds underwent significantproliferation at the tumor site (166-fold higher peak photon countrelative to injected T cells on day 8, P<0.0001, FIGS. 4A, 4B) andsubstantially reduced KPC tumor growth (FIGS. 4A, 4C). However, eventhough they more than doubled the survival of treated mice, Tcell-loaded scaffolds failed to completely clear the disease as all miceeventually developed Rae-1 low/negative immune-escape variants (FIG.4E).

Combined release of CAR-T cells and STING agonist from scaffolds resultsin synergistic maturation and activation of host antigen-presentingcells. The observations described above indicate that targeting a singleantigen with CAR-T cells is unlikely to protect against the outgrowth ofantigen-negative cells—even when the tumor is saturated with optimallystimulated anti-cancer T cells delivered from an implantable scaffold.Accordingly, it was next sought to synergistically launch host T cellresponses in an effort to eliminate residual CAR-resistant tumor celltypes (see FIG. 5A). An intact immune system is able to generateeffective tumor-specific responses, but it requires stimulation to doso. Unfortunately, tumors inhibit the maturation and activation ofantigen-presenting cells located in their draining lymph nodes, andthereby prevent tumor-reactive T cells from differentiating intocytolytic effectors. In an effort to reverse this suppression, thescaffolds were used to achieve high local concentrations of theimmune-stimulatory STING agonist cyclic-di-GMP (c-diGMP) in order torender the tumor milieu more conducive for T cell priming via therecruitment and stimulation of antigen-presenting cells (FIG. 5B).Dendritic cells (DCs), defined by their high expression of the majorhistocompatibility complex class II (MHC-II) and T cell costimulatorymolecules (e.g., CD86), are the most potent antigen-presenting cells andare capable of orchestrating an adaptive anti-tumor immune response.Immune phenotyping of tumor-associated lymph nodes associated withestablished KPC tumors revealed that less than 6% of their DCs(recognized in flow cytometry as CD11c+CD11b+) were appropriatelyactivated, as evidenced by their expression of CD86; the majority of theDCs were tolerogenic (CD86-negative; FIG. 6A). Releasing only c-diGMPfrom implanted scaffolds upregulated CD86 and MHC-II expression by alarge proportion of these DCs, and increased their overall frequency inthe draining lymph nodes 38-fold (FIG. 6B). Following implantation ofCAR-T cell-loaded scaffolds fabricated without the STING agonist,numbers of CD11c+CD86+ mature DCs increased only modestly (9.4-fold),but the MHC-II expression levels on these cells was more than twice ashigh compared to the c-diGMP treatment group (FIG. 6A, 6B). Release ofboth c-diGMP and CAR-T cells from implanted matrices produced asynergistic activation of DCs, reflected by robust increases in thefrequencies of activated DCs (3.7-fold higher compared to c-diGMPalone). Notably, these cells expressed high levels of costimulatorymolecules as well as MHC-II, indicating that they can efficientlycross-present tumor antigens and launch anti-tumor T cell responses(FIG. 6B).

Combined CAR-T cell/STING agonist therapy primes robust tumor-specifichost lymphocyte responses. To measure activation of tumor-specific Tcells in the host, a KPC pancreatic tumor model that expresses thelymphocytic choriomeningitis virus (LCMV) glycoprotein gp33 was used. Asa surrogate pancreatic tumor antigen, this protein enabled use of flowcytometry to analyze how the scaffolds affect the frequency oftetramer-positive cells among CD8+ T cells. In order to distinguishbetween scaffold-delivered and endogenous T cells, the donor cells weregenetically tagged with a CD45.2 marker and CD45.1-transgenic mice wereused as hosts. Mice were treated with biomaterial scaffolds engineeredto release either c-diGMP, CAR-T cells, or a combination of the two intothe tumor. Control mice received no treatment. As expected, spontaneousanti-tumor T cell responses rarely occurred in untreated mice (FIG. 7A),establishing that, despite their expression of the gp33 xenoantigen,KPC-gp33 tumors continue to be highly immunosuppressive. Treatment withc-diGMP-loaded scaffolds or those prepared with lymphocytes aloneproduced host antitumor T cell activities, although the overall responsewas modest (1.3-fold and 2-fold increased numbers in peripheral bloodgp33 tetramer-positive T cells, respectively; FIGS. 7A, 7B). Bycontrast, the combination of c-diGMP and CAR-expressing T cells elicitedsynergistic antitumor responses, which were on average 6.4-fold higherthan implants releasing the lymphocytes only (FIGS. 7A, 7B).

Scaffolds can trigger host antitumor immunity sufficient to clear tumorsand eliminate metastases. To measure the anti-tumor benefits provided byscaffolds that co-deliver the STING agonist along with CAR-programmed Tcells, mice bearing orthotopic KPC tumors were treated using scaffoldsfunctionalized with either c-diGMP alone, or with both c-diGMP and CAR-Tcells; control mice received no treatment. Tumor growth was quantifiedby serial bioluminescence imaging. It was found that the combinedrelease of CAR-T cells and c-diGMP from scaffolds eradicated KPC tumorsin four of ten treated mice (FIGS. 8A-8C), and the other six showedsubstantial tumor regression with an average 37-day improvement insurvival. Although the STING agonist alone never produced completeclearance, it increased survival by 6 days compared to the controlgroup. To determine if the scaffolds elicit global antitumor immunity,the four mice that experienced complete tumor regression (FIGS. 9A, 9B)were re-challenged with a systemic dose of 10⁴ luciferase-expressing KPCtumor cells; tumor-naïve mice were used as controls. Bioluminescenceimaging was then used to quantify differences in growth rates of lungmetastases between the treatment groups. The results establish that allmice cured with CAR-T cell/STING agonist immunotherapy were fullyprotected from this re-challenge, with no measurable tumor mass 4 weeksafter the KPC cells were administered. By contrast, control animalsquickly formed metastatic foci in their lungs and rapidly succumbed totheir disease (FIG. 9B). Thus, appropriately formulated combinationtherapy using CAR-programmed T cells and STING agonists can eliminatelocal tumors and trigger systemic host antitumor immunity powerfulenough to prevent untreated distant metastases.

This work demonstrates a new concept in cancer therapy that achievesboth rapid tumor clearance and systemic antitumor immunity. Thedeveloped scaffolds enable surgeons to deliver cancer-fightinglymphocytes along with potent STING agonists directly to tumors at highlocal concentrations and over an extended period of time. This will notonly maximize treatment successes following, for example, pancreatictumor surgery, but will also reduce healthcare costs because a singletreatment with the disclosed scaffolds is likely to spare patients fromcomplicated second or third surgeries, costly extended hospital stays,cycles of radiation and chemotherapy, and expensive palliative care.More broadly, this platform can eventually shift the focus frombroad-impact chemical and radiation-based approaches to tumor-specificimmunotherapeutics by providing surgeons with an appropriate tool toeasily and safely apply treatments directly to a solid tumor.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient or component.As used herein, the transition term “comprise” or “comprises” meansincludes, but is not limited to, and allows for the inclusion ofunspecified elements, steps, ingredients, or components, even in majoramounts. The transitional phrase “consisting of” excludes any element,step, ingredient or component not specified. The transition phrase“consisting essentially of” limits the scope of the embodiment to thespecified elements, steps, ingredients or components and to those thatdo not materially affect the embodiment. As used herein, a materialeffect would cause a statistically-significant reduction in theanti-tumor effects of a claimed scaffold or method in at least twomeasures of anti-tumor activity disclosed herein.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the following examples or when applicationof the meaning renders any construction meaningless or essentiallymeaningless. In cases where the construction of the term would render itmeaningless or essentially meaningless, the definition should be takenfrom Webster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

1. A lymphocyte scaffold comprising (i) genetically-reprogrammedlymphocytes disposed within a scaffold matrix comprising thin filmnitinol (TFN) micromesh, (ii) a lymphocyte-adhesion moiety comprisingfibrin, and (iii) lymphocyte-activating moieties comprising antibodiesspecific for CD137.
 2. The lymphocyte scaffold of claim 1, wherein thegenetically-reprogrammed lymphocytes comprise T cells and thelymphocyte-activating moieties comprise antibodies specific for CD3,CD28 and CD137.
 3. The lymphocyte scaffold of claim 1, wherein thegenetically-reprogrammed lymphocytes comprise natural killer (NK) cellsand the lymphocyte-activating moieties comprise interleukin 15 andantibodies specific for CD137.
 4. The lymphocyte scaffold of claim 1,wherein the genetically-reprogrammed lymphocytes comprise T cells and NKcells and the lymphocyte-activating moieties comprise antibodiesspecific for CD137.
 5. The lymphocyte scaffold of claim 1, furthercomprising a STING agonist.
 6. The lymphocyte scaffold of claim 5,wherein the STING agonist comprises c-diGMP, c-diAMP, c-GAMP, c-AIMP,(3′,2′)c-AIMP, (2′,2′)c-AIMP, (2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP),c-(dAMP-2′FdIMP), c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP),c-[2′FdAMP(S)-2′FdIMP(S)], c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/orDMXAA.
 7. The lymphocyte scaffold of claim 6, wherein the STING agonistcomprises c-diGMP.
 8. A lymphocyte scaffold comprising (i)genetically-reprogrammed lymphocytes disposed within a scaffold matrixcomprising a micropatterned metallic thin film, and (ii) alymphocyte-activating moiety.
 9. The lymphocyte scaffold of claim 8,further comprising a STING agonist.
 10. The lymphocyte scaffold of claim9, wherein the STING agonist comprises c-diGMP, c-diAMP, c-GAMP, c-AIMP,(3′,2′)c-AIMP, (2′,2′)c-AIMP, (2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP),c-(dAMP-2′FdIMP), c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP),c-[2′FdAMP(S)-2′FdIMP(S)], c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/orDMXAA.
 11. The lymphocyte scaffold of claim 10, wherein the STINGagonist comprises c-diGMP.
 12. The lymphocyte scaffold of claim 11,further comprising a drug eluting polymer.
 13. The lymphocyte scaffoldof claim 12, wherein the STING agonist is embedded within the drugeluting polymer.
 14. The lymphocyte scaffold of claim 12, wherein thedrug eluting polymer comprises PLGA.
 15. The lymphocyte scaffold ofclaim 8, wherein the lymphocytes are T-cells and/or natural killercells.
 16. The lymphocyte scaffold of claim 8, wherein the lymphocytesare CD8+ T-cells.
 17. The lymphocyte scaffold of claim 8, comprising atleast 2×10⁶ lymphocytes.
 18. The lymphocyte scaffold of claim 8,comprising at least 7×10⁶ lymphocytes.
 19. The lymphocyte scaffold ofclaim 8, wherein the lymphocyte-activating moiety comprises at least oneof IL-15, or an antibody specific for CD3, CD28, or CD137.
 20. Thelymphocyte scaffold of claim 8, wherein the lymphocyte-activating moietycomprises antibodies specific for CD3, CD128, and CD137.
 21. Thelymphocyte scaffold of claim 8, wherein the lymphocyte-activating moietycomprises IL-15 and CD137.
 22. The lymphocyte scaffold of claim 8,further comprising an immune stimulant.
 23. The lymphocyte scaffold ofclaim 22, wherein the immune stimulant is a cytokine, an antibody, asmall molecule, an siRNA, a plasmid DNA, and/or a vaccine adjuvant. 24.The lymphocyte scaffold of claim 22, wherein the immune stimulant isselected from (i) a Toll-like receptor ligand selected from CpG, Cpg-28,Poly(I:C), α-galactoceramide, MPLA, VTX-2337, EMD1201081) imiquimod,MGN1703, G100, CBLB502, Hiltonol, and Imiquimod, and/or (ii)17-dimethylaminoethylamino-17-demethoxygeldanamycin).
 25. The lymphocytescaffold of claim 22, wherein the immune stimulant is embedded within adrug eluting polymer.
 26. The lymphocyte scaffold of claim 8, furthercomprising a lymphocyte-adhesion moiety.
 27. The lymphocyte scaffold ofclaim 26, wherein the lymphocyte-adhesion moiety and thelymphocyte-activating moiety are covalently linked.
 28. The lymphocytescaffold of claim 26, wherein the lymphocyte-adhesion moiety comprisesfibrin.
 29. The lymphocyte scaffold of claim 26, wherein thelymphocyte-adhesion moiety comprises a peptide that binds α₁β₁ integrin,α2β₁ integrin, α4β₁ integrin, α5β₁ integrin, or lymphocyte functionassociated antigen (LFA-1).
 30. The lymphocyte scaffold of claim 26,wherein the lymphocyte-adhesion moiety comprises a GFOGER (SEQ ID NO: 1)peptide.
 31. The lymphocyte scaffold of claim 26, wherein thelymphocyte-adhesion moiety comprises a GFOGER (SEQ ID NO: 1) peptide ofSEQ ID NO: 1 or SEQ ID NO:
 2. 32. The lymphocyte scaffold of claim 26,wherein the lymphocyte-adhesion moiety comprises an ICAM-1 peptide. 33.The lymphocyte scaffold of claim 26, wherein the lymphocyte-adhesionmoiety comprises an ICAM-1 peptide of SEQ ID NO:
 3. 34. The lymphocytescaffold of claim 26 wherein the lymphocyte-adhesion moiety comprises aFNIII₁₇₋₁₀ peptide.
 35. The lymphocyte scaffold of claim 26, wherein thelymphocyte-adhesion moiety comprises a FNIII₁₇₋₁₀ peptide of SEQ ID NO:4.
 36. The lymphocyte scaffold of claim 9, wherein the STING agonistand/or the lymphocyte-activating moiety is linked to the scaffoldmatrix.
 37. The lymphocyte scaffold of claim 9, wherein the STINGagonist and/or the lymphocyte-activating moiety is embedded within thescaffold matrix.
 38. The lymphocyte scaffold of claim 9, wherein theSTING agonist and/or the lymphocyte-activating moiety is within abioactive coating overlaying at least a portion of the surface of thescaffold matrix.
 39. The lymphocyte scaffold of claim 38, wherein thebioactive coating comprises a drug eluting polymer.
 40. The lymphocytescaffold of claim 39, further comprising a lymphocyte-adhesion moiety,wherein: the STING agonist is within the drug eluting polymer, the drugeluting polymer forms a monolayer on the surface of the scaffold matrix,and the lymphocyte-adhesion moiety directly coats the drug elutingpolymer.
 41. The lymphocyte scaffold of claim 9, wherein the STINGagonist and/or the lymphocyte-activating moiety is linked to particles.42. The lymphocyte scaffold of claim 41, wherein the particles arelinked to the scaffold and/or embedded within the scaffold matrix. 43.The lymphocyte scaffold of claim 9, wherein the STING agonist and/or thelymphocyte-activating moiety is bound to a liposome of a protocell. 44.The lymphocyte scaffold of claim 43, wherein the ratio of the protocellsto the lymphocytes within the scaffold matrix is 0.5:1; 1:1; 5:1; or10:1.
 45. The lymphocyte scaffold of claim 43, wherein the lymphocytescaffold comprises 7×10⁶ to 1×10¹⁰ protocells.
 46. The lymphocytescaffold of claim 8, wherein the micropatterned metallic thin filmcomprises a TFN micromesh.
 47. A lymphocyte scaffold consisting of ascaffold matrix, genetically reprogrammed lymphocytes, and threelymphocyte-activating moieties.
 48. The lymphocyte scaffold of claim 47,wherein the scaffold matrix is an alginate scaffold, a collagen/alginatescaffold, a chitosan scaffold, a self-assembling peptide scaffold, amesoporous silica scaffold, a micropatterned metallic thin filmscaffold, or a PLGA scaffold.
 49. The lymphocyte scaffold of claim 48,wherein the micropatterned metallic thin film scaffold is a TFNmicromesh scaffold.
 50. The lymphocyte scaffold of claim 47, wherein thescaffold matrix is an alginate scaffold.
 51. The lymphocyte scaffold ofclaim 47, wherein the scaffold matrix is a polymeric calciumcross-linked alginate scaffold.
 52. The lymphocyte scaffold of claim 47,wherein the genetically reprogrammed lymphocytes are geneticallyreprogrammed T-cells and/or natural killer cells.
 53. The lymphocytescaffold of claim 47, wherein the genetically reprogrammed lymphocytesare CD8+ T-cells.
 54. The lymphocyte scaffold of claim 47, comprising atleast 2×10⁶ genetically reprogrammed lymphocytes.
 55. The lymphocytescaffold of claim 47, comprising at least 7×10⁶ genetically reprogrammedlymphocytes.
 56. The lymphocyte scaffold of claim 47, wherein thelymphocyte-activating moieties comprise antibodies specific for CD3,CD28, and CD137.
 57. The lymphocyte scaffold of claim 47, wherein thelymphocyte-activating moieties are linked to the scaffold.
 58. Thelymphocyte scaffold of claim 47, wherein the lymphocyte-activatingmoieties are embedded within the scaffold.
 59. A lymphocyte scaffoldcomprising: a scaffold matrix, genetically-reprogrammed lymphocytes, anda lymphocyte-activating moiety.
 60. The lymphocyte scaffold of claim 59,further comprising a STING agonist.
 61. The lymphocyte scaffold of claim60, wherein the STING agonist comprises c-diGMP, c-diAMP, c-GAMP,c-AIMP, (3′,2′)c-AIMP, (2′,2′)c-AIMP, (2′,3′)c-AIMP, c-AIMP(S),c-(dAMP-dIMP), c-(dAMP-2′FdIMP), c-(2′FdAMP-2′FdIMP),(2′,3′)c-(AMP-2′FdIMP), c-[2′FdAMP(S)-2′FdIMP(S)],c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/or DMXAA.
 62. The lymphocytescaffold of claim 60, wherein the STING agonist comprises c-diGMP. 63.The lymphocyte scaffold of claim 59, wherein thegenetically-reprogrammed lymphocytes are genetically-reprogrammedT-cells and/or natural killer cells.
 64. The lymphocyte scaffold ofclaim 59, wherein the genetically-reprogrammed lymphocytes are CD8+T-cells.
 65. The lymphocyte scaffold of claim 59, comprising at least7×10⁶ genetically-reprogrammed lymphocytes.
 66. The lymphocyte scaffoldof claim 59, wherein the lymphocyte-activating moiety comprisesantibodies specific for CD3, CD28, and/or CD137.
 67. The lymphocytescaffold of claim 59, further comprising an immune stimulant.
 68. Thelymphocyte scaffold of claim 67, wherein the immune stimulant is acytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and/ora vaccine adjuvant.
 69. The lymphocyte scaffold of claim 67, wherein theimmune stimulant is selected from (i) a Toll-like receptor ligandselected from CpG, Cpg-28, Poly(I:C), α-galactoceramide, MPLA, VTX-2337,EMD1201081) imiquimod, MGN1703, G100, CBLB502, Hiltonol, and Imiquimod,and/or (ii) 17-dimethylaminoethylamino-17-demethoxygeldanamycin). 70.The lymphocyte scaffold of claim 59, further comprising alymphocyte-adhesion moiety.
 71. The lymphocyte scaffold of claim 70,wherein the lymphocyte-adhesion moiety comprises fibrin.
 72. Thelymphocyte scaffold of claim 70, wherein the lymphocyte-adhesion moietycomprises a peptide that binds α₁β₁ integrin, α2β₁ integrin, α4β₁integrin, α5β₁ integrin, or lymphocyte function associated antigen(LFA-1).
 73. The lymphocyte scaffold of claim 70, wherein thelymphocyte-adhesion moiety comprises a GFOGER (SEQ ID NO: 1) peptide.74. The lymphocyte scaffold of claim 70, wherein the lymphocyte-adhesionmoiety comprises a GFOGER (SEQ ID NO: 1) peptide of SEQ ID NO: 1 or SEQID NO:
 2. 75. The lymphocyte scaffold of claim 70, wherein thelymphocyte-adhesion moiety comprises an ICAM-1 peptide.
 76. Thelymphocyte scaffold of claim 70, wherein the lymphocyte-adhesion moietycomprises an ICAM-1 peptide of SEQ ID NO:
 3. 77. The lymphocyte scaffoldof any claim 70, wherein the lymphocyte-adhesion moiety comprises aFNIII₁₇₋₁₀ peptide.
 78. The lymphocyte scaffold of claim 70, wherein thelymphocyte-adhesion moiety comprises a FNIII₁₇₋₁₀ peptide of SEQ ID NO:4.
 79. The lymphocyte scaffold of claim 60, wherein the STING agonistand/or the lymphocyte-activating moiety is linked to the scaffoldmatrix.
 80. The lymphocyte scaffold of claim 60, wherein the STINGagonist and/or the lymphocyte-activating moiety is embedded within thescaffold matrix.
 81. The lymphocyte scaffold of claim 60, wherein theSTING agonist and/or the lymphocyte-activating moiety is within abioactive coating overlaying at least a portion of the surface of thescaffold matrix.
 82. The lymphocyte scaffold of claim 60, wherein theSTING agonist and/or the lymphocyte-activating moiety is linked toparticles.
 83. The lymphocyte scaffold of claim 82, wherein theparticles are linked to the scaffold matrix and/or embedded within thescaffold matrix.
 84. The lymphocyte scaffold of claim 60, wherein theSTING agonist and/or the lymphocyte-activating moiety is bound to aliposome of a protocell.
 85. The lymphocyte scaffold of claim 84,wherein the ratio of the protocells to the lymphocytes within thescaffold matrix is 0.5:1; 1:1; 5:1; or 10:1.
 86. The lymphocyte scaffoldof claim 59, wherein the lymphocyte scaffold comprises 7×10⁶ to 1×10¹⁰protocells.
 87. The lymphocyte scaffold of claim 59, wherein thescaffold matrix is an alginate scaffold, a collagen/alginate scaffold, achitosan scaffold, a self-assembling peptide scaffold, a mesoporoussilica scaffold, a micropatterned metallic thin film scaffold, or a PLGAscaffold.
 88. The lymphocyte scaffold of claim 87, wherein themicropatterned metallic thin film scaffold is a TFN micromesh scaffold.89. The lymphocyte scaffold of claim 59, wherein the scaffold matrix isan alginate scaffold.
 90. The lymphocyte scaffold of claim 59, whereinthe scaffold matrix is a polymeric calcium cross-linked alginatescaffold.
 91. A lymphocyte scaffold comprising: (i) a scaffold matrixcomprising an alginate scaffold; (ii) genetically-reprogrammedlymphocytes; (iii) a lymphocyte-adhesion moiety comprising a GFOGER (SEQID NO: 1) peptide; (iv) lymphocyte-activating moieties comprisingantibodies specific for CD3, CD28, and CD137; and (v) a STING agonist.92. The lymphocyte scaffold of claim 91, wherein the STING agonistcomprises c-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP,(2′,2′)c-AIMP, (2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP),c-(dAMP-2′FdIMP), c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP),c-[2′FdAMP(S)-2′FdIMP(S)], c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/orDMXAA.
 93. The lymphocyte scaffold of claim 92, wherein the STINGagonist comprises c-diGMP.
 94. The lymphocyte scaffold of claim 91,wherein the genetically-reprogrammed lymphocytes aregenetically-reprogrammed T-cells and/or natural killer cells.
 95. Thelymphocyte scaffold of claim 91, wherein the genetically-reprogrammedlymphocytes are CD8+ T-cells.
 96. The lymphocyte scaffold of claim 91,comprising at least 7×10⁶ genetically-reprogrammed lymphocytes.
 97. Thelymphocyte scaffold of claim 91, further comprising an immune stimulant.98. The lymphocyte scaffold of claim 97, wherein the immune stimulant isa cytokine, an antibody, a small molecule, an siRNA, a plasmid DNA,and/or a vaccine adjuvant.
 99. The lymphocyte scaffold of claim 97,wherein the immune stimulant is selected from (i) a Toll-like receptorligand selected from CpG, Cpg-28, Poly(I:C), α-galactoceramide, MPLA,VTX-2337, EMD1201081) imiquimod, MGN1703, G100, CBLB502, Hiltonol, andImiquimod, and/or (ii)17-dimethylaminoethylamino-17-demethoxygeldanamycin).
 100. Thelymphocyte scaffold of claim 91, wherein the GFOGER (SEQ ID NO: 1)peptide comprises a peptide of SEQ ID NO: 1 or SEQ ID NO:
 2. 101. Thelymphocyte scaffold of claim 91, wherein the STING agonist and/or thelymphocyte-activating moieties are linked to the scaffold matrix. 102.The lymphocyte scaffold of claim 91, wherein the STING agonist and/orthe lymphocyte-activating moieties are embedded within the scaffoldmatrix.
 103. The lymphocyte scaffold of claim 91, wherein the STINGagonist and/or the lymphocyte-activating moieties are within a bioactivecoating overlaying at least a portion of the surface of the scaffoldmatrix.
 104. The lymphocyte scaffold of claim 91, wherein the STINGagonist and/or the lymphocyte-activating moieties are linked toparticles.
 105. The lymphocyte scaffold of claim 104, wherein theparticles are linked to the scaffold matrix and/or embedded within thescaffold matrix.
 106. The lymphocyte scaffold of claim 91, wherein theSTING agonist and/or the lymphocyte-activating moieties are bound to aliposome of a protocell.
 107. The lymphocyte scaffold of claim 106,wherein the ratio of the protocells to the lymphocytes within thescaffold matrix is 0.5:1; 1:1; 5:1; or 10:1.
 108. The lymphocytescaffold of claim 91, wherein the lymphocyte scaffold comprises 7×10⁶ to1×10¹⁰ protocells.
 109. The lymphocyte scaffold of claim 91, wherein thealginate scaffold is a polymeric calcium cross-linked alginate scaffold.110. A lymphocyte scaffold comprising: (i) a scaffold matrix material,(ii) natural killer cells with anti-cancer activity, and (iii)lymphocyte-activating moieties comprising IL-15 and an antibody specificto CD137.
 111. The lymphocyte scaffold of claim 110, wherein thescaffold matrix material comprises a micropatterned metallic thin film.112. The lymphocyte scaffold of claim 111, wherein the micropatternedmetallic thin film comprises a TFN micromesh.
 113. The lymphocytescaffold of claim 110, further comprising a STING agonist.
 114. Thelymphocyte scaffold of claim 112, wherein the STING agonist comprisesc-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP, (2′,2′)c-AIMP,(2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP), c-(dAMP-2′FdIMP),c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP), c-[2′FdAMP(S)-2′FdIMP(S)],c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/or DMXAA.
 115. The lymphocytescaffold of claim 113, further comprising a drug-eluting polymer. 116.The lymphocyte scaffold of claim 115, wherein the drug eluting polymercomprises PLGA.
 117. The lymphocyte scaffold of claim 115, wherein theSTING agonist is embedded within a drug eluting polymer.
 118. Thelymphocyte scaffold of claim 110, further comprising alymphocyte-adhesion moiety.
 119. The lymphocyte scaffold of claim 118,wherein the lymphocyte-adhesion moiety comprises fibrin.
 120. Thelymphocyte scaffold of claim 118, wherein the lymphocyte-adhesion moietycomprises a peptide that binds α₁β₁ integrin, α2β₁ integrin, α4β₁integrin, α5β₁ integrin, or lymphocyte function associated antigen(LFA-1).
 121. The lymphocyte scaffold of claim 118, wherein thelymphocyte-adhesion moiety comprises a GFOGER (SEQ ID NO: 1) peptide.122. The lymphocyte scaffold of claim 118, wherein thelymphocyte-adhesion moiety comprises a GFOGER (SEQ ID NO: 1) peptide ofSEQ ID NO: 1 or SEQ ID NO:
 2. 123. The lymphocyte scaffold of claim 118,wherein the lymphocyte-adhesion moiety comprises an ICAM-1 peptide. 124.The lymphocyte scaffold of claim 118, wherein the lymphocyte-adhesionmoiety comprises an ICAM-1 peptide of SEQ ID NO:
 3. 125. The lymphocytescaffold of any claim 118, wherein the lymphocyte-adhesion moietycomprises a FNIII₁₇₋₁₀ peptide.
 126. The lymphocyte scaffold of claim118, wherein the lymphocyte-adhesion moiety comprises a FNIII₁₇₋₁₀peptide of SEQ ID NO:
 4. 127. A method of treating a heterogenous solidtumor comprising escape variant tumor cells in a subject comprisingimplanting a lymphocyte scaffold of claim 59 into the subject within aproximity of the heterogenous solid tumor cell sufficient to lead to thedestruction of the heterogenous solid tumor in the subject, therebytreating the heterogenous solid tumor comprising escape variant tumorcells.
 128. A method of vaccinating a subject against development ofcancer recurrence comprising implanting a lymphocyte scaffold of claim59 into the subject within a proximity of a heterogenous solid tumor orwithin a solid tumor resection bed in the subject, thereby vaccinatingthe subject against development of cancer recurrence.
 129. A method oftreating tumor cells in a subject in need thereof comprising implantinga lymphocyte scaffold of claim 59 into the subject within a tumorresection bed thereby treating the tumor cells in the subject.
 130. Themethod of claim 127, wherein a treated tumor cell is an adrenal cancercell, a brain cancer cell, a breast cancer cell, a cervical cancer cell,a colon cancer cell, a colorectal cancer cell, an ear, nose and throat(ENT) cancer cell, an endometrial cancer cell, an esophageal cancercell, a gastrointestinal cancer cell, a glioma cell, a head and neckcancer cell, an intestinal cancer cell, a kidney cancer cell, a livercancer cell, a lung cancer cell, a lymph node cancer cell, a melanomacell, a neuroblastoma cell, an ovarian cancer cell, a pancreatic cancercell, a prostate cancer cell, a rectal cancer cell, a seminoma cell, askin cancer cell, a stomach cancer cell, a teratoma cell, a thyroidcancer cell, or a uterine cancer cell.
 131. The method of claim 127,wherein a treated tumor cell is a glioblastoma cell, a pancreaticadenocarcinoma cell, or an ovarian cancer cell.
 132. A method ofreducing surgical treatment failure caused by metastatic relapse afterresection of a primary tumor, comprising administering a lymphocytescaffold of claim 59 to a tumor resection bed of a subject therebyreducing surgical treatment failure caused by metastatic relapse afterprimary tumor resection.
 133. The method of claim 132, wherein theprimary tumor comprises a seminoma cell, a melanoma cell, a teratomacell, a neuroblastoma cell, a glioma cell, a rectal cancer cell, anendometrial cancer cell, a kidney cancer cell, an adrenal cancer cell, athyroid cancer cell, a skin cancer cell, a brain cancer cell, a cervicalcancer cell, an intestinal cancer cell, a liver cancer cell, a coloncancer cell, a stomach cancer cell, a head and neck cancer cell, agastrointestinal cancer cell, a lymph node cancer cell, an esophagealcancer cell, a colorectal cancer cell, a pancreatic cancer cell, an ear,nose and throat (ENT) cancer cell, a breast cancer cell, a prostatecancer cell, a uterine cancer cell, an ovarian cancer cell, or a lungcancer cell.
 134. A method of treating a subject for cancer comprisingimplanting into the subject a medical device coated with a lymphocytescaffold comprising a TFN micromesh, genetically-modified lymphocytes,and a lymphocyte activating moiety.
 135. The method of claim 134,wherein the implanting comprises a minimally invasive procedure. 136.The method of claim 134, wherein the medical device comprises a stent.137. A kit to form a lymphocyte scaffold to treat a solid tumor in asubject comprising (i) a scaffold matrix; and (ii) lymphocyte-activatingmoieties comprising antibodies specific for CD3, CD28, and CD137. 138.The kit of claim 137, further comprising porous particles.
 139. The kitof claim 137, further comprising liposomes.
 140. The kit of claim 137,further comprising protocells.
 141. The kit of claim 137, furthercomprising a STING agonist.
 142. The kit of claim 141, wherein the STINGagonist comprises c-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP,(2′,2′)c-AIMP, (2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP),c-(dAMP-2′FdIMP), c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP),c-[2′FdAMP(S)-2′FdIMP(S)], c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/orDMXAA.
 143. The kit of claim 141, wherein the STING agonist comprisesc-diGMP.
 144. The kit of claim 137, further comprising an immunestimulant.
 145. The kit of claim 144, wherein the immune stimulant is acytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and/ora vaccine adjuvant.
 146. The kit of claim 144, wherein the immunestimulant is selected from (i) a Toll-like receptor ligand selected fromCpG, Cpg-28, Poly(I:C), α-galactoceramide, MPLA, VTX-2337, EMD1201081)imiquimod, MGN1703, G100, CBLB502, Hiltonol, and Imiquimod, and/or (ii)17-dimethylaminoethylamino-17-demethoxygeldanamycin).
 147. The kit ofclaim 137, further comprising a lymphocyte-adhesion moiety.
 148. The kitof claim 147, wherein the lymphocyte-adhesion moiety comprises fibrin.149. The kit of claim 147, wherein the lymphocyte-adhesion moietycomprises a peptide that binds α₁β₁ integrin, α2β₁ integrin, α4β₁integrin, α5β₁ integrin, or lymphocyte function associated antigen(LFA-1).
 150. The kit of claim 147, wherein the lymphocyte-adhesionmoiety comprises a GFOGER (SEQ ID NO: 1) peptide.
 151. The kit of claim147, wherein the lymphocyte-adhesion moiety comprises a GFOGER (SEQ IDNO: 1) peptide of SEQ ID NO: 1 or SEQ ID NO:
 2. 152. The kit of claim147, wherein the lymphocyte-adhesion moiety comprises an ICAM-1 peptide.153. The kit of claim 147, wherein the lymphocyte-adhesion moietycomprises an ICAM-1 peptide of SEQ ID NO:
 3. 154. The kit of claim 147,wherein the lymphocyte-adhesion moiety comprises a FNIII₇₋₁₀ peptide.155. The kit of claim 147, wherein the lymphocyte-adhesion moietycomprises a FNIII₇₋₁₀ peptide of SEQ ID NO:
 4. 156. The kit of claim137, wherein the kit comprises 7×10⁶ to 1×10¹⁰ particles, liposomes orprotocells.
 157. The kit of claim 137, further comprisinggenetically-reprogrammed lymphocytes.
 158. The kit of claim 157, whereinthe genetically-reprogrammed lymphocytes are T-cells and/or naturalkiller cells.
 159. The kit of claim 157, wherein thegenetically-reprogrammed lymphocytes are CD8+ T-cells.
 160. The kit ofclaim 157, wherein the lymphocytes comprise at least 7×10⁶ lymphocytes.161. The kit of claim 137, wherein the scaffold matrix comprisesalginate, collagen, chitosan, a self-assembling peptide, mesoporoussilica, TFN micromesh, or PLGA.
 162. The kit of claim 161, wherein thescaffold matrix comprises alginate.
 163. The kit of claim 137, furthercomprising calcium.
 164. The kit of claim 137, further comprising a drugeluting polymer.
 165. The kit of claim 164, wherein the drug elutingpolymer is coated on the scaffold matrix.
 166. The kit of claim 164,wherein a STING agonist or an immune stimulant is embedded within thedrug eluting polymer.
 167. The kit of claim 164, wherein the drugeluting polymer comprises PLGA.
 168. A kit to form a lymphocyte scaffoldcomprising: (i) a scaffold matrix comprising TFN micromesh (ii) alymphocyte-adhesion moiety comprising fibrin; and (iii)lymphocyte-activating moieties comprising antibodies specific for CD3,CD28, and CD137.
 169. The kit of claim 168, further comprising a vectorfor genetically reprogramming lymphocytes.
 170. The kit of claim 168,wherein the fibrin and the lymphocyte-activating moieties are covalentlylinked.
 171. The kit of claim 168, further comprisinggenetically-reprogrammed lymphocytes.
 172. The kit of claim 171, whereinthe genetically-reprogrammed lymphocytes are T-cells and/or naturalkiller cells.
 173. The kit of claim 171, wherein thegenetically-reprogrammed lymphocytes are CD8+ T-cells.
 174. The kit ofclaim 171, wherein the lymphocytes comprise at least 7×10⁶ lymphocytes.175. The kit of claim 168, further comprising a STING agonist and/or animmune stimulant.
 176. The kit of claim 175, wherein the STING agonistcomprises c-diGMP, c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP,(2′,2′)c-AIMP, (2′,3′)c-AIMP, c-AIMP(S), c-(dAMP-dIMP),c-(dAMP-2′FdIMP), c-(2′FdAMP-2′FdIMP), (2′,3′)c-(AMP-2′FdIMP),c-[2′FdAMP(S)-2′FdIMP(S)], c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/orDMXAA.
 177. The kit of claim 175, wherein the STING agonist comprisesc-diGMP.
 178. The kit of claim 175, wherein the immune stimulantcomprises a cytokine, an antibody, a small molecule, an siRNA, a plasmidDNA, and/or a vaccine adjuvant.
 179. The kit of claim 175 furthercomprising a drug eluting polymer, wherein the STING agonist and/or theimmune stimulant are embedded within the drug eluting polymer.
 180. Akit to form a lymphocyte scaffold comprising: (i) a scaffold matrixcomprising an alginate scaffold, (ii) a lymphocyte-adhesion moietycomprising a GFOGER (SEQ ID NO: 1) peptide, (iii) lymphocyte-activatingmoieties comprising antibodies specific for CD3, CD28, and CD137, and(iv) a STING agonist.
 181. The kit of claim 180, further comprisingporous particles.
 182. The kit of claim 180, further comprisingliposomes.
 183. The kit of claim 180, further comprising protocells.184. The kit of claim 180, wherein the STING agonist comprises c-diGMP,c-diAMP, c-GAMP, c-AIMP, (3′,2′)c-AIMP, (2′,2′)c-AIMP, (2′,3′)c-AIMP,c-AIMP(S), c-(dAMP-dIMP), c-(dAMP-2′FdIMP), c-(2′FdAMP-2′FdIMP),(2′,3′)c-(AMP-2′FdIMP), c-[2′FdAMP(S)-2′FdIMP(S)],c-[2′FdAMP(S)-2′FdIMP(S)](POM)2, and/or DMXAA.
 185. The kit of claim180, wherein the STING agonist comprises c-diGMP.
 186. The kit of claim180, further comprising an immune stimulant.
 187. The kit of claim 186,wherein the immune stimulant is a cytokine, an antibody, a smallmolecule, an siRNA, a plasmid DNA, and/or a vaccine adjuvant.
 188. Thekit of claim 186, wherein the immune stimulant is selected from (i) aToll-like receptor ligand selected from CpG, Cpg-28, Poly(I:C),α-galactoceramide, MPLA, VTX-2337, EMD1201081) imiquimod, MGN1703, G100,CBLB502, Hiltonol, and Imiquimod, and/or (ii)17-dimethylaminoethylamino-17-demethoxygeldanamycin).
 189. The kit ofclaim 180, wherein the kit comprises 7×10⁶ to 1×10¹⁰ particles,liposomes or protocells.
 190. The kit of claim 180, further comprising avector for genetically reprogrammed lymphocytes.
 191. The kit of claim180, further comprising genetically-reprogrammed lymphocytes.
 192. Thekit of claim 191, wherein the genetically-reprogrammed lymphocytes areT-cells and/or natural killer cells.
 193. The kit of claim 191, whereinthe genetically-reprogrammed lymphocytes are CD8+ T-cells.
 194. The kitof claim 191, wherein the lymphocytes comprise at least 7×10⁶lymphocytes.
 195. The kit of claim 180, further comprising calcium. 196.An implantable medical device comprising: (i) a micropatterned metallicthin film scaffold, (ii) genetically reprogrammed lymphocytes, and (iii)a lymphocyte-activating moiety.
 197. The implantable medical device ofclaim 196 further comprising a STING agonist and/or an immune stimulant.198. The implantable medical device of claim 196 further comprising adrug eluting polymer, wherein the STING agonist and/or the immunestimulant is embedded within the drug eluting polymer.
 199. Theimplantable medical device of claim 196, wherein the micropatternedmetallic thin film scaffold has a three-dimensional shape.
 200. Theimplantable medical device of claim 199, wherein the three-dimensionalshape is a cylinder.
 201. The implantable medical device of claim 196,comprising a stent.
 202. The implantable medical device of claim 1967,comprising a minimally invasive medical device.
 203. The implantablemedical device of claim 196, wherein the micropatterned metallic thinfilm scaffold is stacked in layers.
 204. The implantable medical deviceof claim 196, wherein the micropatterned metallic thin film comprises aTFN micromesh.
 205. The implantable medical device of claim 196, whereinthe genetically-reprogrammed lymphocytes are at a concentration of atleast 7×10⁶ cells/cm³.